Maleamic Acid Polymer Derivatives and Their Bioconjugates

ABSTRACT

The present invention is directed to maleamic acid derivatives of water soluble polymers, to chemically stable water-soluble polymer succinamic acid-active agent conjugates, and to methods for reproducibly preparing, characterizing and using such polymer reagents and their conjugates.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 11/904,990, filed Sep. 27, 2007, which is adivisional application of U.S. patent application Ser. No. 10/750,996,filed Dec. 31, 2003, now U.S. Pat. No. 7,329,721, which claims thebenefit of priority to U.S. provisional application Ser. No. 60/468,340,filed May 5, 2003, and to U.S. provisional application Ser. No.60/437,251, filed Dec. 31, 2002, the disclosures of each of theforegoing are incorporated herein by reference in their entireties.

FIELD OF THE INVENTION

This invention relates generally to the field of polymer chemistry, andmore specifically to chemically stable active agent conjugates preparedfrom maleimide- or maleamic acid functionalized water-soluble polymerssuch as polyethylene glycol, and to methods for synthesizing,characterizing, and using such polymer reagents and conjugates.

BACKGROUND OF THE INVENTION

Due to recent advances in biotechnology, therapeutic proteins and otherbiomolecules, e.g. antibodies and antibody fragments, can now beprepared on a large scale, making such biomolecules more widelyavailable. Unfortunately, the clinical usefulness of potentialtherapeutic biomolecules is often hampered by their rapid proteolyticdegradation, low bioavailability, instability upon manufacture, storageor administration, or by their immunogenicity. Due to the continuedinterest in administering proteins and other biomolecules fortherapeutic use, various approaches to overcoming these deficiencieshave been explored.

One approach that has been widely explored is the modification ofproteins and other potentially therapeutic molecules by covalentattachment of a water-soluble polymer such as polyethylene glycol or“PEG” (Abuchowski, A., et al, J. Biol. Chem. 252 (11), 3579 (1977);Davis, S., et al., Clin. Exp Immunol., 46, 649-652 (1981). Thebiological properties of PEG-modified proteins, also referred to asPEG-conjugates or pegylated proteins, have been shown, in many cases, tobe considerably improved over those of their non-pegylated counterparts(Herman, et al., Macromol. Chem. Phys., 195, 203-209 (1994).Polyethylene glycol-modified proteins have been shown to possess longercirculatory times in the body due to increased resistance to proteolyticdegradation, and also to possess increased thermostability (Abuchowski,A., et al., J. Biol. Chem., 252, 3582-3586 (1977). A similar increase inbioefficacy is observed with other biomolecules, e.g. antibodies andantibody fragments (Chapman, A., Adv. Drug Del. Rev, 54, 531-545(2002)).

Typically, attachment of polyethylene glycol to a drug or other surfaceis accomplished using an activated PEG derivative, that is to say, a PEGhaving at least one activated terminus suitable for reaction with anucleophilic center of a biomolecule (e.g., lysine, cysteine and similarresidues of proteins). Most commonly employed are methods based upon thereaction of an activated PEG with protein amino groups, such as thosepresent in the lysine side chains of proteins. Polyethylene glycolhaving activated end groups suitable for reaction with the amino groupsof proteins include PEG-aldehydes (Harris, J. M., Herati, R. S., PolymPrepr. (Am. Chem. Soc., Div. Polym. Chem.), 32(1), 154-155 (1991), mixedanhydrides, N-hydroxysuccinimide esters, carbonylimadazolides, andchlorocyanurates (Herman, S., et al., Macromol. Chem. Phys. 195, 203-209(1994)). Although many proteins have been shown to retain activityduring PEG modification, in some instances, polymer attachment throughprotein amino groups can be undesirable, such as when derivatization ofspecific lysine residues inactivates the protein (Suzuki, T., et al.,Biochimica et Biophysica Acta 788, 248-255 (1984)). Moreover, since mostproteins possess several available/accessible amino groups, the polymerconjugates formed are typically mixtures of mono-pegylated,di-pegylated, tri-pegylated species and so on, which can be difficultand also time-consuming to characterize and separate. Further, suchmixtures are often not reproducibly prepared, which can create problemsduring scale-up for regulatory approval and subsequentcommercialization.

One method for avoiding these problems is to employ a site-selectivepolymer reagent that targets functional groups other than amines. Oneparticularly attractive target is the thiol group, which in proteins inpresent in the amino acid, cysteine. Cysteines are typically lessabundant in proteins than lysines, thus reducing the likelihood ofprotein deactivation upon conjugation to these thiol-containing aminoacids. Moreover, conjugation to cysteine sites can often be carried outin a well-defined manner, leading to the formation of single speciespolymer-conjugates.

Polyethylene glycol derivatives having a thiol-selective reactive endgroup include maleimides, vinyl sulfones, iodoacetamides, thiols, anddisulfides, with maleimides being the most popular. These derivativeshave all been used for coupling to the cysteine side chains of proteins(Zalipsky, S. Bioconjug. Chem. 6, 150-165 (1995); Greenwald, R. B. etal. Crit. Rev. Ther. Drug Carrier Syst. 17, 101-161 (2000); Herman, S.,et al., Macromol. Chem. Phys. 195, 203-209 (1994)). However, many ofthese reagents have not been widely exploited due to the difficulty intheir synthesis and purification.

Polyethylene glycol derivatives having a terminal maleimide group areone of the most popular types of sulfhydryl-selective reagents, and arecommercially available from a number of sources. Although not widelyappreciated, or recognized, the Applicants have recognized that manyPEG-maleimides unfortunately exhibit hydrolytic instability duringstorage and/or conjugation to a drug candidate. More particularly, asubstantial degree of hydrolysis of the maleimide ring has beenobserved, both prior to and after conjugation. This instability canresult in the formation of multiple species of drug conjugates within adrug-conjugate composition. The various drug conjugate species arelikely to possess similar biological activities, but may differ in theirpharmacokinetic properties. This is particularly disadvantageous forcompositions intended for patient administration, since the resultingdrug compositions can be ill-defined mixtures of drug conjugate specieswhose particular safety and accumulation profiles are unknown. Moreover,due to different factors impacting hydrolysis rates, inconsistencybetween drug conjugate batch compositions can present an additionalproblem.

Another problem has been observed by the applicants is the de-pegylationof conjugates prepared from PEG maleimides to yield mixtures of altereddrug and detached PEG impurity. For these reasons, the Applicants havefound that PEG maleimides can be undesirable reagents for coupling tothiol groups on target drugs or other active agents. Previous attemptsto address this problem have focused on increasing the stability of apolymer maleimide by making it less prone to hydrolysis (i.e.,ring-opening). See for example, U.S. Patent Application Publication No.US 2003/0065134.

Thus, the applicants have realized a continuing need in the art for thedevelopment of new activated PEGs useful for coupling to biologicallyactive molecules, desirably in a site-selective fashion, that overcomethe shortcomings of presently-available thiol-selective polymer reagentsand are stable during both storage and coupling. This invention meetsthose needs.

SUMMARY OF THE INVENTION

The present invention provides thiol-selective polymer reagents andtheir conjugates that (i) are stable during storage and coupling, (ii)are resistant to hydrolysis, and (iii) exhibit increased resistance tode-pegylation, thereby allowing formation of substantially chemicallystable and well-defined drug conjugate compositions to be described ingreater detail below.

The present invention is based upon the Applicants' recognition of theneed for an alternative to conventional polymer maleimide reagents. Inresponse to this need, the Applicants have devised an approach that iscompletely contrary to other approaches employed to date. That is tosay, rather than utilizing a customary approach and attempting toprevent hydrolysis of the maleimide ring, the Applicants have insteadintentionally forced open the maleimide ring, to provide polymerreagents and conjugates where the “maleimide” is converted to its stablesuccinamic acid opened-ring form.

More particularly, in one aspect, provided herein is a method wherein amaleimide group of a water-soluble polymer is forcibly (intentionally)converted to its ring-open maleamic acid form, either prior to or moreconventionally after coupling to an active agent. In this way, amaleamic acid or succinamic acid polymer composition is provided thatpossesses: (i) well-defined components, and (ii) a diminished tendencytowards hydrolysis, particularly in comparison to its maleimide-derived,succinimide counterparts.

More specifically, in one aspect, the invention provides a method forpreparing a polymer conjugate. The method includes the steps of (a)providing a water-soluble polymer comprising a maleimide group, and (b)reacting the polymer with an active agent that possesses a nucleophileunder conditions effective to couple the active agent to the watersoluble polymer via a Michael-type addition reaction to form apolymer-succinimide-linked active agent conjugate. This conjugate, instep (c), is then treated under conditions effective to force open thesuccinimide ring, to form a polymer-succinamic acid-conjugate.

In one embodiment, the maleimide ring is forced open via a hydrolysisreaction. Typically, the ring-opening hydrolysis is carried out in thepresence of a base that can be in solution or on a solid support.Typical pHs for conducting the hydrolysis are in a range of about 6 to12.

In a preferred embodiment, the hydrolysis is carried out underconditions effective to provide a chemically stable polymer-succinamicacid-conjugate composition.

In one embodiment of the method, the hydrolysis reaction is carried outuntil at least about 15% of the polymer succinamic acid conjugate isformed, based upon the conversion of the closed ring-form. Inalternative embodiments, the hydrolysis reaction is carried out until atleast about 35%, or 50%, or 80%, or 95%, or 98% or essentially 100%polymer succinamic acid conjugate is formed, i.e., where the polymermaleimide conjugate is essentially fully ring-opened.

Water soluble polymers for use in the invention include poly(alkyleneoxide), poly(vinyl pyrrolidone), poly(vinyl alcohol), polyoxazoline,poly(acryloylmorpholine), and poly(oxyethylated polyol). A preferredwater-soluble polymer is polyethylene glycol.

In yet another aspect, provided herein is a polymer succinamic acidconjugate composition prepared by the method described above.

In yet another aspect, provided herein is a composition that comprises:

where POLY is a water-soluble polymer segment, L is an optional linker,and “Nu-Active agent” represents an active agent comprising anucleophile, “Nu”. Preferred nucleophiles include thiol, thiolate, andamino.

In yet another aspect, the invention provides a protein derivatized witha water-soluble polymer, where the polymer is coupled to the protein viasuccinimide groups covalently attached to either cysteine sulfyhydrylgroups or lysine amino groups, and substantially all of the succinimidegroups are present in a ring-opened form.

These and other objects and features of the invention will become morefully apparent when read in conjunction with the following figures anddetailed description.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates an exemplary reaction of both a polymer maleimide anda polymer maleamic acid with a thiol group of a representative activeagent, in this case, a protein, to form a polymer-succinamic acidconjugate of the invention;

FIG. 2 illustrates an exemplary reaction of both a polymer maleimide anda polymer maleamic acid with an amino group of a representative activeagent, in this case, a protein, to form a polymer-succinamic acidconjugate of the invention;

FIG. 3 is a plot of the logarithm of the concentration of anillustrative branched polymer linkered maleimide over time as describedin detail in Example 2.

DETAILED DESCRIPTION OF THE INVENTION

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to the particularpolymers, synthetic techniques, active agents, and the like as such mayvary. It is also to be understood that the terminology used herein isfor describing particular embodiments only, and is not intended to belimiting.

In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions describedbelow.

DEFINITIONS

The following terms as used herein have the meanings indicated.

As used in the specification, and in the appended claims, the singularforms “a”, “an”, “the”, include plural referents unless the contextclearly dictates otherwise.

“PEG” or “poly(ethylene glycol)” as used herein, is meant to encompassany water-soluble poly(ethylene oxide). Typically, PEGs for use in thepresent invention will comprise one of the two following structures:“—(CH₂CH₂O)_(n)—” or “—(CH₂CH₂O)_(n-1)CH₂CH₂—,” depending upon whetheror not the terminal oxygen(s) has been displaced, e.g., during asynthetic transformation. The variable (n) ranges from 3 to 3000, andthe terminal groups and architecture of the overall PEG may vary. WhenPEG further comprises a linker moiety (to be described in greater detailbelow), the atoms comprising the linker, when covalently attached to aPEG segment, do not result in formation of (i) an oxygen-oxygen bond(—O—O—, a peroxide linkage), or (ii) a nitrogen-oxygen bond (N—O, O—N).“PEG” means a polymer that contains a majority, that is to say, greaterthan 50%, of subunits that are —CH₂CH₂O—. PEGs for use in the inventioninclude PEGs having a variety of molecular weights, structures orgeometries (e.g., branched, linear, forked PEGs, dendritic, and thelike), to be described in greater detail below.

“PEG diol”, also known as alpha-,omega-dihydroxylpoly(ethylene glycol),can be represented in brief form as HO-PEG—OH, where PEG is as definedabove.

“Water-soluble”, in the context of a polymer of the invention or a“water-soluble polymer segment” is any segment or polymer that issoluble in water at room temperature. Typically, a water-soluble polymeror segment will transmit at least about 75%, more preferably at leastabout 95% of light, transmitted by the same solution after filtering. Ona weight basis, a water-soluble polymer or segment thereof willpreferably be at least about 35% (by weight) soluble in water, morepreferably at least about 50% (by weight) soluble in water, still morepreferably about 70% (by weight) soluble in water, and still morepreferably about 85% (by weight) soluble in water. It is most preferred,however, that the water-soluble polymer or segment is about 95% (byweight) soluble in water or completely soluble in water.

An “end-capping” or “end-capped” group is an inert or non-reactive grouppresent on a terminus of a polymer such as PEG. An end-capping group isone that does not readily undergo chemical transformation under typicalsynthetic reaction conditions. An end capping group is generally analkoxy group, —OR, where R is an organic radical comprised of 1-20carbons and is preferably lower alkyl (e.g., methyl, ethyl) or benzyl.“R” may be saturated or unsaturated, and includes aryl, heteroaryl,cyclo, heterocyclo, and substituted forms of any of the foregoing. Forinstance, an end capped PEG will typically comprise the structure“RO—(CH₂CH₂O)_(n)—”, where R is as defined above. Alternatively, theend-capping group can also advantageously comprise a detectable label.When the polymer has an end-capping group comprising a detectable label,the amount or location of the polymer and/or the moiety (e.g., activeagent) to which the polymer is coupled, can be determined by using asuitable detector. Such labels include, without limitation, fluorescers,chemiluminescers, moieties used in enzyme labeling, colorimetric (e.g.,dyes), metal ions, radioactive moieties, and the like. The end-cappinggroup can also advantageously comprise a phospholipid. When the polymerhas an end-capping group such as a phospholipid, unique properties (suchas the ability to form organized structures with similarly end-cappedpolymers) are imparted to the polymer. Exemplary phospholipids include,without limitation, those selected from the class of phospholipidscalled phosphatidylcholines. Specific phospholipids include, withoutlimitation, those selected from the group consisting ofdilauroylphosphatidylcholine, dioleylphosphatidylcholine,dipalmitoylphosphatidylcholine, disteroylphosphatidylcholine,behenoylphosphatidylcholine, arachidoylphosphatidylcholine, andlecithin.

“Non-naturally occurring” with respect to a polymer of the inventionmeans a polymer that in its entirety is not found in nature. Anon-naturally occurring polymer of the invention may however contain oneor more subunits or segments of subunits that are naturally occurring,so long as the overall polymer structure is not found in nature.

“Molecular mass” in the context of a water-soluble polymer of theinvention such as PEG, refers to the nominal average molecular mass of apolymer, typically determined by size exclusion chromatography, lightscattering techniques, or intrinsic velocity determination in1,2,4-trichlorobenzene. The polymers of the invention are typicallypolydisperse, possessing low polydispersity values of less than about1.20.

The term “reactive” or “activated” refers to a functional group thatreacts readily or at a practical rate under conventional conditions oforganic synthesis. This is in contrast to those groups that either donot react or require strong catalysts or impractical reaction conditionsin order to react (i.e., a “nonreactive” or “inert” group).

“Not readily reactive” or “inert” with reference to a functional grouppresent on a molecule in a reaction mixture, indicates that the groupremains largely intact under conditions effective to produce a desiredreaction in the reaction mixture.

A “protecting group” is a moiety that prevents or blocks reaction of aparticular chemically reactive functional group in a molecule undercertain reaction conditions. The protecting group will vary dependingupon the type of chemically reactive group being protected as well asthe reaction conditions to be employed and the presence of additionalreactive or protecting groups in the molecule. Functional groups whichmay be protected include, by way of example, carboxylic acid groups,amino groups, hydroxyl groups, thiol groups, carbonyl groups and thelike. Representative protecting groups for carboxylic acids includeesters (such as a p-methoxybenzyl ester), amides and hydrazides; foramino groups, carbamates (such as tert-butoxycarbonyl) and amides; forhydroxyl groups, ethers and esters; for thiol groups, thioethers andthioesters; for carbonyl groups, acetals and ketals; and the like. Suchprotecting groups are well-known to those skilled in the art and aredescribed, for example, in T. W. Greene and G. M. Wuts, ProtectingGroups in Organic Synthesis, Third Edition, Wiley, New York, 1999, andreferences cited therein.

A functional group in “protected form” refers to a functional groupbearing a protecting group. As used herein, the term “functional group”or any synonym thereof is meant to encompass protected forms thereof.

The term “linker” is used herein to refer to an atom or a collection ofatoms optionally used to link interconnecting moieties, such as apolymer segment and a maleimide. The linkers of the invention aregenerally hydrolytically stable.

A “physiologically cleavable” or “hydrolyzable” or “degradable” bond isa relatively weak bond that reacts with water (i.e., is hydrolyzed)under physiological conditions. The tendency of a bond to hydrolyze inwater will depend not only on the general type of linkage connecting twocentral atoms but also on the substituents attached to these centralatoms. Appropriate hydrolytically unstable or weak linkages include butare not limited to carboxylate ester, phosphate ester, anhydrides,acetals, ketals, acyloxyalkyl ether, imines, orthoesters, peptides andoligonucleotides, thioesters, thiolesters, and carbonates.

An “enzymatically degradable linkage” means a linkage that is subject todegradation by one or more enzymes.

A “hydrolytically stable” linkage or linker, for the purposes of thepresent invention, and in particular in reference to the polymers of theinvention, refers to an atom or to a collection of atoms, that ishydrolytically stable under normal physiological conditions. That is tosay, a hydrolytically stable linkage does not undergo hydrolysis underphysiological conditions to any appreciable extent over an extendedperiod of time. Examples of hydrolytically stable linkages include butare not limited to the following: carbon-carbon bonds (e.g., inaliphatic chains), ethers, amides, urethanes, amines, and the like.Hydrolysis rates of representative chemical bonds can be found in moststandard chemistry textbooks.

“Branched” in reference to the geometry or overall structure of apolymer refers to polymer having 2 or more polymer “arms”. A branchedpolymer may possess 2 polymer arms, 3 polymer arms, 4 polymer arms, 6polymer arms, 8 polymer arms or more. One particular type of highlybranched polymer is a dendritic polymer or dendrimer, that for thepurposes of the invention, is considered to possess a structure distinctfrom that of a branched polymer.

“Branch point” refers to a bifurcation point comprising one or moreatoms at which a polymer splits or branches from a linear structure intoone or more additional polymer arms.

A “dendrimer” is a globular, size monodisperse polymer in which allbonds emerge radially from a central focal point or core with a regularbranching pattern and with repeat units that each contribute a branchpoint. Dendrimers exhibit certain dendritic state properties such ascore encapsulation, making them unique from other types of polymers.

“Substantially” or “essentially” means nearly totally or completely, forinstance, 95% or greater of some given quantity.

An “alkyl” or “alkylene” group, depending upon its position in amolecule and the number of points of attachment of the group to atomsother than hydrogen, refers to a hydrocarbon chain or moiety, typicallyranging from about 1 to 20 atoms in length. Such hydrocarbon chains arepreferably but not necessarily saturated unless so indicated and may bebranched or straight chain, although typically straight chain ispreferred. Exemplary alkyl groups include methyl, ethyl, propyl, butyl,pentyl, 1-methylbutyl, 1-ethylpropyl, 3-methylpentyl, and the like.

“Lower alkyl” or “lower alkylene” refers to an alkyl or alkylene groupas defined above containing from 1 to 6 carbon atoms, and may bestraight chain or branched, as exemplified by methyl, ethyl, n-butyl,i-butyl, t-butyl.

“Cycloalkyl” or “cycloalkylene”, depending upon its position in amolecule and the number of points of attachment to atoms other thanhydrogen, refers to a saturated or unsaturated cyclic hydrocarbon chain,including polycyclics such as bridged, fused, or spiro cyclic compounds,preferably made up of 3 to about 12 carbon atoms, more preferably 3 toabout 8.

“Lower cycloalkyl” or “lower cycloalkylene” refers to a cycloalkyl groupcontaining from 1 to 6 carbon atoms.

“Alicyclic” refers to any aliphatic compound that contains a ring ofcarbon atoms. An alicyclic group is one that contains a “cycloalkyl” or“cycloalkylene” group as defined above that is substituted with one ormore alkyl or alkylenes.

“Non-interfering substituents” are those groups that, when present in amolecule, are typically non-reactive with other functional groupscontained within the molecule.

The term “substituted” as in, for example, “substituted alkyl,” refersto a moiety (e.g., an alkyl group) substituted with one or morenon-interfering substituents, such as, but not limited to: C₃-C₈cycloalkyl, e.g., cyclopropyl, cyclobutyl, and the like; halo, e.g.,fluoro, chloro, bromo, and iodo; cyano; alkoxy, lower phenyl;substituted phenyl; and the like. For substitutions on a phenyl ring,the substituents may be in any orientation (i.e., ortho, meta, or para).

“Alkoxy” refers to an —O—R group, wherein R is alkyl or substitutedalkyl, preferably C₁-C₂₀ alkyl (e.g., methyl, ethyl, propyl, benzyl,etc.), preferably C₁-C₇.

As used herein, “alkenyl” refers to a branched or unbranched hydrocarbongroup of 1 to 15 atoms in length, containing at least one double bond,such as ethenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl,octenyl, decenyl, tetradecenyl, and the like.

The term “alkynyl” as used herein refers to a branched or unbranchedhydrocarbon group of 2 to 15 atoms in length, containing at least onetriple bond, ethynyl, n-propynyl, isopropynyl, n-butynyl, isobutynyl,octynyl, decynyl, and so forth.

“Aryl” means one or more aromatic rings, each of 5 or 6 core carbonatoms. Aryl includes multiple aryl rings that may be fused, as innaphthyl or unfused, as in biphenyl. Aryl rings may also be fused orunfused with one or more cyclic hydrocarbon, heteroaryl, or heterocyclicrings. As used herein, “aryl” includes heteroaryl.

“Heteroaryl” is an aryl group containing from one to four heteroatoms,preferably N, O, or S, or a combination thereof. Heteroaryl rings mayalso be fused with one or more cyclic hydrocarbon, heterocyclic, aryl,or heteroaryl rings.

“Heterocycle” or “heterocyclic” means one or more rings of 5-12 atoms,preferably 5-7 atoms, with or without unsaturation or aromatic characterand having at least one ring atom which is not a carbon. Preferredheteroatoms include sulfur, oxygen, and nitrogen.

“Substituted heteroaryl” is heteroaryl having one or morenon-interfering groups as substituents.

“Substituted heterocycle” is a heterocycle having one or more sidechains formed from non-interfering substituents.

“Electrophile” refers to an ion, atom, or collection of atoms that maybe ionic, having an electrophilic center, i.e., a center that iselectron seeking, capable of reacting with a nucleophile.

“Nucleophile” refers to an ion or atom or collection of atoms that maybe ionic, having a nucleophilic center, i.e., a center that is seekingan electrophilic center, and capable of reacting with an electrophile.

“Active agent” as used herein includes any agent, drug, compound,composition of matter or mixture which provides some pharmacologic,often beneficial, effect that can be demonstrated in-vivo or in vitro.This includes foods, food supplements, nutrients, nutriceuticals, drugs,vaccines, antibodies, vitamins, and other beneficial agents. As usedherein, these terms further include any physiologically orpharmacologically active substance that produces a localized or systemiceffect in a patient.

“Pharmaceutically acceptable excipient” or “pharmaceutically acceptablecarrier” refers to an excipient that can be included in the compositionsof the invention and that causes no significant adverse toxicologicaleffects to the patient.

“Pharmacologically effective amount,” “physiologically effectiveamount,” and “therapeutically effective amount” are used interchangeablyherein to mean the amount of a PEG-active agent conjugate present in apharmaceutical preparation that is needed to provide a desired level ofactive agent and/or conjugate in the bloodstream or in the targettissue. The precise amount will depend upon numerous factors, e.g., theparticular active agent, the components and physical characteristics ofpharmaceutical preparation, intended patient population, patientconsiderations, and the like, and can readily be determined by oneskilled in the art, based upon the information provided herein andavailable in the relevant literature.

“Multi-functional” in the context of a polymer of the invention means apolymer backbone having 3 or more functional groups contained therein,where the functional groups may be the same or different, and aretypically present on the polymer termini. Multi-functional polymers ofthe invention will typically contain from about 3-100 functional groups,or from 3-50 functional groups, or from 3-25 functional groups, or from3-15 functional groups, or from 3 to 10 functional groups, or willcontain 3, 4, 5, 6, 7, 8, 9 or 10 functional groups within the polymerbackbone.

A “difunctional” polymer means a polymer having two functional groupscontained therein, typically at the polymer termini. When the functionalgroups are the same, the polymer is said to be homodifunctional. Whenthe functional groups are different, the polymer is said to beheterobifunctional

A basic or acidic reactant described herein includes neutral, charged,and any corresponding salt forms thereof.

“Polyolefinic alcohol” refers to a polymer comprising an olefin polymerbackbone, such as polyethylene, having multiple pendant hydroxyl groupsattached to the polymer backbone. An exemplary polyolefinic alcohol ispolyvinyl alcohol.

As used herein, “non-peptidic” refers to a polymer backbonesubstantially free of peptide linkages. However, the polymer may includea minor number of peptide linkages spaced along the repeat monomersubunits, such as, for example, no more than about 1 peptide linkage perabout 50 monomer units.

The term “patient,” refers to a living organism suffering from or proneto a condition that can be prevented or treated by administration of apolymer of the invention, typically but not necessarily in the form of apolymer-active agent conjugate, and includes both humans and animals.

“Optional” or “optionally” means that the subsequently describedcircumstance may or may not occur, so that the description includesinstances where the circumstance occurs and instances where it does not.

By “residue” is meant the portion of a molecule remaining after reactionwith one or more molecules. For example, a biologically active moleculeresidue in a polymer conjugate of the invention typically corresponds tothe portion of the biologically active molecule up to but excluding thecovalent linkage resulting from reaction of a reactive group on thebiologically active molecule with a reactive group on a polymer reagent.

The term “conjugate” is intended to refer to the entity formed as aresult of covalent attachment of a molecule, e.g., a biologically activemolecule or any reactive surface, to a reactive polymer molecule,preferably a reactive poly(ethylene glycol).

The term “electron withdrawing group” refers to a chemical moiety thatbrings electron density towards itself and away from other areas of amolecule through either mesomeric mechanisms (i.e., adding or removinglocal electron density through π bonds) or inductive mechanisms (i.e.,an electronegative moiety withdrawing electron density along a σ bond,thereby polarizing the bond).

“Chemically stable” in the context of the compositions, polymers andconjugates described herein, refers to a sample that undergoes a 5% orless change in its polymer composition (that is to say, the subjectpolymer, conjugate or the like is not chemically altered or degraded inany significant manner, for example, where applicable, by de-pegylationor hydrolysis to result in chemical species that are different inamounts or in their structure from those originally present in thesample) over a 3 month time period when measured from the time ofinitial sample preparation and stored as a buffered solution atsubstantially neutral pHs (e.g., 6.8 to 7.2) under ambient conditions.

A polymer or composition that is “resistant to hydrolysis”, in thecontext of the present invention, is one that undergoes hydrolysis to anextent less than 5%, when stored over a 3 month time period whenmeasured from the time of initial sample preparation, and stored as abuffered solution at substantially neutral pHs (e.g., 6.8 to 7.2) underambient conditions.

A “2 or 3-substituted succinamic acid” refers to the position of asubstituent, e.g., a nucleophile that is part of an active agent on apolymer succinamic acid, where the carboxylic acid group of thesuccinamic acid represents carbon number 1, and the carbon or positionadjacent to that is carbon number 2, and so on.

Overview of the Invention

Customarily, maleimide groups positioned on a polymer are used tocovalently attach or conjugate a polymer to an active agent such as abiomolecule, especially a biomolecule containing one or more reactivethiol groups. Such thiol groups may be naturally occurring, oralternatively, the biomolecule may be modified or engineered to containa thiol suitable for coupling to a maleimide. Under certain morerigorous reaction conditions, e.g., at higher pH levels, active aminogroups on a biomolecule can also add to a maleimide group on a polymerderivative to form the corresponding conjugate. Through a series ofexperiments, the Applicants have recognized that certainpolymer-maleimide derivatives, depending upon their structure, are pronetowards hydrolysis to form the ring-opened maleamic acid form of thepolymer, either before or after conjugation to an active agent. Thehydrolysis reaction is not only dependent upon the overall structure ofthe polymer derivative, but is also pH dependent. Generally, the rate ofhydrolysis increases with increasing pH. Additionally, depending uponthe moisture content and pH of the resulting composition, formation ofthe ring-open form of the polymer conjugate can also occur upon storageof a dry polymer conjugate composition, e.g., one where the active agentis a non-protein drug. In cases where ring opening occurs, the resultingcomposition may actually be a complicated mixture of ring-open andring-closed conjugates. In general, such hydrolysis can be problematic,particularly for commercial pharmaceutical compositions where long-termstability and consistency in drug lots are highly desirable features.

In an effort to address this problem, the invention provides certainmaleamic acid polymer derivatives, their conjugates, and compositionscontaining them, along with methods for making and using such maleamicacid-derived polymer derivatives. The polymers of the invention areprovided to overcome the problems associated withmaleimide-functionalized polymers by forcing or promoting the hydrolysisof the maleimide ring, either before or more preferably subsequent toconjugation. In this way, ring-open polymer maleamic acid structures areprovided which are much more stable than their maleimide (orsuccinimide) counterparts. Preferably, the polymer maleamic acidcompositions of the invention possess well-defined and substantiallyunchanging amounts of polymer maleamic acid or polymer succinamic acidconjugates, such that the compositions of the invention are particularlywell-suited for use as pharmaceutical compositions for administration tomammalian subjects.

Two illustrative reaction schemes demonstrating an overview of thisapproach are provided herein as FIG. 1 and FIG. 2. Reaction Scheme I(FIG. 1) illustrates the reaction of both a polymer-maleimide (structureI) and a polymer maleamic acid (structure II) with a thiol-group of abiologically active molecule, in this case, a protein. The reactionconditions shown in FIGS. 1 and 2 are meant to be exemplary only and arenot meant to be limiting. Reaction Scheme II (FIG. 2) similarlyillustrates reaction of both a polymer-maleimide and a polymer maleamicacid with an amino-group of a biologically active molecule, in thiscase, a protein. In each scheme, both of the isomeric structures of theconjugated succinamic acid products are shown (structures IV and V,where IV-A and V-A correspond to the thiol-conjugated polymer succinamicacid and IV-B and V-B correspond to the amino-conjugated polymersuccinamic acid. The two different products arise from addition of theincoming nucleophile to either of the two carbons, C-2 or C-3, of thedouble bond of the maleimide ring.

In looking at either FIG. 1 or FIG. 2, it can be seen that whileconjugation of an active agent to a polymer maleamic acid, II, can becarried out, the reaction is particularly slow. For this reason, a morepreferred route to the desired succinamic acid conjugate is byhydrolysis of the polymer succinimide conjugate, shown generally asstructure III. That is to say, in comparison to the correspondingpolymer maleimide derivatives, maleamic acid polymer derivatives areless reactive with nucleophiles to form the corresponding conjugates.Thus, conjugation to a polymer maleimide followed by ring opening isgenerally preferred over ring-opening of a polymer maleimide followed byconjugation, although both approaches result in formation ofpolymer-succinamic acid conjugates, shown generally as structures IV andV.

Formation of Maleamic and Succinamic Acid Polymer Derivatives andConjugates

Polymer Maleimides.

In general, the methods provided herein begin with a polymer maleimide.Polymer maleimides can be obtained from commercial sources, such as fromNektar, Huntsville, Ala. For instance, polymer maleimides such asmPEG(MAL)₂, mPEG2(MAL)₂, mPEG2-MAL, and mPEG-MAL are commerciallyavailable from Nektar in a wide range of molecular weights. Structurescorresponding to these polymer maleimides are found in the NektarCatalog, 2001, entitled, “Polyethylene Glycol and Derivatives forBiomedical Applications”, on page 8, and are incorporated herein byreference.

Alternatively, the polymer maleimides of the invention can be preparedby any of a number of synthetic routes including the following. In oneapproach, a maleimide-terminated polymer is prepared by reacting afunctional group attached to a polymer segment (i.e., an activatedpolymer segment) with a functional group of a bifunctional reagenthaving as one of its functional groups either a maleimide or afunctional group that can be converted to a maleimide, such as an aminogroup. Reacting the polymer segment with a bifunctional reagent resultsin covalent attachment, typically through a hydrolytically stablelinkage, of the reagent to the polymer segment to provide either apolymer maleimide or a polymer maleimide precursor.

For example, the bifunctional reagent may possess the structure A-L-B,wherein A is a first functional group that is reactive with a secondfunctional group on the polymer segment to form a linkage, L, to formPOLY-L-B, where B is a maleimide or a functional group that can bereadily converted to a maleimide (e.g., an amine that can be convertedto a maleimide by reaction with methoxycarbonylmaleimide). In the aboveapproach, A can be any of a number of functional groups such as halo,hydroxyl, active ester such as N-succinimidyl ester, active carbonate,acetal, aldehyde, aldehyde hydrate, alkenyl, acrylate, methacrylate,acrylamide, active sulfone, thiol, carboxylic acid, isocyanate,isothiocyanate, maleimide, vinylsulfone, dithiopyridine, vinylpyridine,iodoacetamide, and epoxide, suitable for reacting with the target groupon the activated polymer reagent.

In instances where an approach is employed that utilizes a polymeramine, POLY-L_(0,1)-NH₂ as a starting material or intermediate, theamine can be transformed into a maleimide, for example, using maleicanhydride. Preferably, the polymer amine is purified prior to conversionto a maleimide group, for example, by chromatography or any othersuitable method, to improve the purity of the final maleimide product.In one particular approach, a polymer amine is first reacted with maleicanhydride to form an open ring amide carboxylic acid intermediate, whichis then closed in a second step by heating the intermediate in thepresence of acetic anhydride and a salt of acetic acid, such as sodiumor potassium acetate. Preferably, the intermediate is heated at atemperature ranging from about 50° C. to about 140° C. for about 0.2 toabout 5 hours.

Alternatively, an amino group on POLY-L_(0,1)-NH₂ can be transformedinto a maleimide by reaction with a reagent such asN-methoxycarbonylmaleimide orexo-7-oxa[2.2.1]bicycloheptane-2,3-dicarboxylic anhydride.

Structures corresponding to representative polymer maleamic acids andpolymer succinamic acid conjugates (provided in the sections thatfollow) can be extended to the corresponding starting materials andintermediates as described above.

Conjugation to an Active Agent

A polymer maleimide is coupled to a biologically active molecule oractive agent using suitable reaction conditions known in the art.Precise conditions will of course vary depending upon the particularactive agent, the precise nucleophile that is to undergo a Michael typeaddition to the maleimide group, the polymer reagent itself and thelike.

Suitable conjugation conditions are those conditions of time,temperature, pH, reagent concentration, solvent, and the like sufficientto effect conjugation between a polymer maleimide and an active agent.The specific conditions depend upon, among other things, the activeagent, the type of conjugation desired, the presence of other materialsin the reaction mixture and so forth. Sufficient conditions foreffecting conjugation in any particular case can be determined by one ofordinary skill in the art upon a reading of the disclosure herein,reference to the relevant literature, and/or through routineexperimentation.

Exemplary conjugation conditions include carrying out the conjugationreaction at a pH of from about 6 to about 10, and at, for example, a pHof about 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10. More preferably,a polymer maleimide is typically conjugated to a sulfhydryl-containingactive agent at pHs ranging from about 6-9.5, more preferably at pHsfrom about 7-9, and even more preferably at pHs from about 7 to 8. Mostpreferably, thiol-selective conjugation is conducted at pHs around 7.

Reaction temperatures are highly dependent on the reactivity of thebiomolecule and can typically range from 0° C. to 75° C., preferablyfrom 10° C. to 45° C., and more preferably from 18° C. to 28° C. Highertemperatures may deactivate the more sensitive biomolecules but may benecessary to convert the more resistant ones.

Conjugation reactions can be carried out in a buffer such as a phosphateor acetate buffer or similar system.

Generally, a slight molar excess of polymer maleimide is employed, forexample, a 1.5 to 15-fold molar excess, preferably a 2-fold to 10 foldmolar excess. The molar ratio of precursor polymer to biologicallyactive molecule can range from 1.0 to 50, preferably from 1.0 to 8.0,and more preferably from 1.04 to 1.5. Exemplary ratios of polymerreagent to active agent include molar ratios of about 1:1 (polymerreagent:active agent), 1.5:1, 2:1, 3:1, 4:1, 5:1, 6:1, 8:1, or 10:1. Theconjugation reaction is allowed to proceed until substantially nofurther conjugation occurs, which can generally be determined bymonitoring the progress of the reaction over time. Progress of thereaction can be monitored by withdrawing aliquots from the reactionmixture at various time points and analyzing the reaction mixture bySDS-PAGE or MALDI-TOF mass spectrometry or any other suitable analyticalmethod. Once a plateau is reached with respect to the amount ofconjugate formed or the amount of unconjugated polymer remaining, thereaction is assumed to be complete.

Again, reaction time is a function of the reactivity of the particularactive agent and becomes longer when the active agent is both slow toreact and sensitive to temperature. In such cases, longer reaction timesaccompanied by moderate reaction temperatures may be required. Typicalreaction times can range from five minutes to 10 days, preferably from30 minutes to 48 hours, and more preferably from 2 to 17 hours, againdependent upon the reactivity of the components, as typically determinedby small scale trial reactions. Agitation (e.g., stirring, shaking,etc.) can optionally be used to facilitate the coupling reaction. Forsterically hindered sulfhydryl groups, required reaction times may besignificantly longer.

Reactions with amino groups proceed at higher pHs, but are relativelyslow in comparison to the reaction with thiol groups.

Particular reaction conditions and methodology should be such that theactive molecule retains at least partial activity.

Conjugates thus prepared can then be further characterized usinganalytical methods such as MALDI, capillary electrophoresis, gelelectrophoresis, and/or chromatography. Polymer conjugates resultingfrom a Michael type addition of an active agent to a polymer maleimideare referred to herein as polymer succinimide conjugates or conjugatedpolymer succinimides (e.g., see structure III).

Maleimide Ring Hydrolysis.

Having a polymer maleimide or a conjugated polymer succinimide in hand(corresponding to structures I and III, respectively), the polymerspecies is then hydrolyzed to its open ring form. When starting with apolymer maleimide, the corresponding opened-ring form is referred toherein as a polymer maleamic acid, corresponding to structure II. Whenderived from a conjugated polymer succinimide (III), the correspondingopened-ring form is referred to herein as a conjugated polymersuccinamic acid, corresponding to structure IV or structure V.Structures IV and V are structural isomers, differing only in the pointof attachment of the nucleophilic group of the active agent. An incomingnucleophile undergoing a Michael type addition reaction to the maleimidecan add either at position C2, relative to the final carboxyl carbon ofthe opened ring form designated as C1, or at position C3.

Generally, a conjugated succinamic acid is prepared by exposing apolymer maleimide, preferably conjugated to an active agent, to aqueousbase under conditions effective to hydrolyze the maleimide group of thepolymer to a measurable degree. Preferably, the hydrolysis reaction iscarried out by adjusting the reaction conditions (amount of water,temperature, relative molar ratios of reactants, etc.) to achieve adesirable extent of hydrolysis or ring opening. Typically, thehydrolysis reaction is carried out to form at least about 15% or greaterof the polymer open-ring form, either conjugated or non-conjugated,relative to its closed-ring polymer counterpart. In focusing now on thepolymer succinamic acid conjugates, particularly preferred compositionsof the invention contain at least about 35%, preferably 40%, 45%, 50%,55% 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or essentially 100%conjugated polymer succinamic acid relative to its unhydrolyzed polymercounterpart. For instance, a hydrolyzed polymer composition thatcomprises 60% polymer succinamic acid conjugate will therefore contain40% conjugated polymer succinimide (its closed ring polymercounterpart).

Most preferably, hydrolysis is carried out until complete, that is tosay, until essentially all of the polymer maleimide or succinimidegroups in the conjugate are converted to their ring-open form and theresulting composition is essentially absent any detectable amounts ofthe closed ring form. Polymer conjugates that are fully ring opened arethe most preferred, since their tendency to undergo the reversereaction, i.e., a dehydrolysis reaction, is minimal under the hydrolysisconditions employed for the forward, ring-opening reaction. Relative tothe partially ring-opened compositions described above, compositionsthat are fully ring-opened are the most stable towards further chemicaltransformations such as depegylation or hydrolysis.

Particularly preferred compositions are those containing less than about50% by weight of the closed ring form, or less than about 40% of theclosed ring form. More preferred are compositions having less than about30%, or more preferably less than about 15% of the closed ring form.Even more preferred are compositions containing less than about 10% byweight, or less than about 5% by weight, or even 2% or less of theclosed ring form.

Turning now to the conditions employed for effecting hydrolysis,hydrolysis is generally conducted under basic conditions. By raising thepH of the reaction mixture or solution above neutral pHs, the ringopening reaction can essentially be forced to completion. To achieve themost efficient (i.e., shortest) reaction times, it is desirable toconduct the hydrolysis at the highest pH possible, e.g., up to about 12,to achieve ring-opening while not adversely impacting the activity orintegrity of the active agent.

Base-promoted ring opening can be carried out using a basic solution ora base bonded to a solid support material, i.e., an ion exchanger.Preferred bases are those that provide the proper pH for a reasonablyrapid ring opening without incurring undesirable side reactions.Exemplary bases include alkali metals such as sodium or potassium metal;alkali metal hydroxides such as lithium hydroxide, sodium hydroxide,potassium hydroxide, and the like, and quaternary ammonium hydroxidessuch as tetraammonium hydroxide, tetrabutylammonium hydroxide, andbenzyltrimethylammonium hydroxide,

The hydrolysis is typically carried out at pHs ranging from about 6 toabout 12. That is to say, the hydrolysis is conducted at a pH selectedfrom about: 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0,11.5, or even 12.0. Preferred pHs range from about 7.5 to 11.

The hydrolysis reaction may optionally include a buffer. Exemplarybuffers include organic buffers such as HEPES, i.e.4-(2-hydroxyethyl)-1-piperzineethanesulfonic acid; as well as bufferssuch as sodium, potassium, or ammonium salts of anions such as citrate,alkylsulfonates, hydroxide, acetate, carbonate, tetraborate,bicarbonate, phosphate, and hydrogen phosphate. Ideally, one should, ona small scale, evaluate the particular base and optional buffer systemwith the particular maleimide or maleimide conjugate prior to carryingout a large process to make certain that the rate of conversion isacceptable and that there are no undesirable side reactions.

Suitable temperatures for effecting hydrolysis range from about 4° C. toabout 75° C., preferably from about 0° C. to about 60° C., morepreferably from about 15° C. to about 45° C., and more preferably fromabout 18° C. to about 30° C. As previously mentioned, reaction times arepH dependent. Reaction times will typically range from about 5 min toseveral days, e.g., 96 hours or more, if no side reactions are evident.However, preferred times are from about 30 min to about 24 hrs, and morepreferably from about 2 hr to about 17 hr. Agitation can optionally beused to facilitate the reaction.

In certain instances, e.g., in the presence of some buffers at certainconcentrations of buffer and polymer maleimide, the ring openingreactions can slow down with time. To attain a stable reaction rate, itmay be desirable to use a buffer system that provides a stable pH overtime under the hydrolysis conditions employed, or alternatively, the pHmay be monitored and base added periodically, if necessary, to maintaina constant pH range. It should be emphasized, however, that a constantpH is not required to obtain complete ring opening.

Ideally, the polymer succinimide conjugate is exposed to a base at asufficient temperature and for a sufficient period of time such that adesired degree of ring opening is achieved. Since the ring-openingreaction can occur over a range of pH values, it is preferable to try tobalance achieving short reaction times, e.g., at the higher pHs, and tofavor a greater extent of hydrolysis, e.g., to form a fully hydrolyzedcomposition where essentially all of the succinimide rings arehydrolyzed, against the possibility of the occurrence of competitiveside reactions that could lead to undesirable mixtures of products ordeactivated active agent. Therefore, through small scale trialreactions, one should ideally choose pH values that minimize suchundesired side reactions. For instance, at pH values between 5.0 and6.5, side reactions are minimal but ring opening of either the polymericmaleimides or their conjugates is very slow, often to a prohibitivedegree.

For example, mPEG2-MAL-40K, Structure VII,

obtainable from Nektar (Huntsville, Ala.), undergoes a very limiteddegree of hydrolysis of the maleimide ring under certain conditions toform the corresponding maleamic acid. Data corresponding to the kineticsof the ring opening reaction is provided in Example 2.

Again, it should be understood that if the polymer derivative isintended for conjugation to a biologically active molecule, thehydrolysis reaction conditions and methodology should be such that thebiologically active molecule retains at least partial activity.

Following hydrolysis, the pH of polymer succinamic acidconjugate-containing reaction mixture is typically adjusted to pHs fromabout 5.5 to 8. The composition is then optionally desalted and dried,for example, by lyophilization. The resulting composition can then befurther purified, is desired, for example by precipitation orchromatography. Different chromatographic separation approaches that canbe utilized include SDS PAGE, gel permeation chromatography, and ionexchange chromatography. One particularly preferred approach is ionexchange chromatography, which is advantageous in separating the polymersuccinamic acid conjugate, having a carboxylic acid functionality, fromthe corresponding closed ring conjugated polymer succinimide. Loweringof the pH and drying of the composition, e.g., by lyophilization, isparticularly advantageous for compositions where the extent of ringopening is not complete, that is to say, where hydrolysis has not yetgone to completion, since lower pHs and the absence of water disfavorfurther hydrolysis. In this way, the composition of the reaction mixtureis essentially “frozen”, i.e., is chemically stable, at a certainnon-equilibrium amount of ring-open form.

Moreover, compositions containing the polymer succinamic acid conjugatesdescribed herein may also be further purified to obtain/isolatedifferent PEGylated succinamic acid species. Alternatively, and morepreferably for lower molecular weight PEGs, e.g., having molecularweights less than about 20 kilodaltons, preferably less than or equal toabout 10 kilodaltons, a product mixture can be purified to obtain adistribution around a certain number of PEGs per protein molecule, whereapplicable. For example, a product mixture can be purified to obtain anaverage of anywhere from one to five PEGs per protein, typically anaverage of about 3 PEGs per protein. The strategy for purification ofthe final conjugate reaction mixture will depend upon a number offactors—the molecular weight of the polymer employed, the particularprotein, the desired dosing regimen, and the residual activity and invivo properties of the individual conjugate(s) species. This approach ismore generally applicable to conjugates prepared by reaction of a PEGmaleimide with protein amino groups that typically are present in agreater abundance within a given protein than are sulhydryl groups.

If desired, PEG conjugates having different molecular weights can beisolated using gel filtration chromatography. While this approach can beused to separate PEG conjugates having different molecular weights, thisapproach is generally ineffective for separating positional isomershaving different pegylation sites within a protein. For example, gelfiltration chromatography can be used to separate from each othermixtures of PEG 1-mers, 2-mers, 3-mers, etc., although each of therecovered PEG-mer compositions may contain PEGs attached to differentreactive amino groups (e.g., lysine residues) within the protein.

Gel filtration columns suitable for carrying out this type of separationinclude Superdex™ and Sephadex™ columns available from AmershamBiosciences. Selection of a particular column will depend upon thedesired fractionation range desired. Elution is generally carried outusing a non-amine based buffer, such as phosphate, acetate, or the like.The collected fractions may be analysed by a number of differentmethods, for example, (i) OD at 280 nm for protein content, (ii) BSAprotein analysis, (iii) iodine testing for PEG content (Sims G. E. C.,et al., Anal. Biochem, 107, 60-63, 1980), or alternatively, (iv) byrunning an SDS PAGE gel, followed by staining with barium iodide.

Separation of positional isomers can be carried out by reverse phasechromatography using, for example, an RP-HPLC C18 column (AmershamBiosciences or Vydac) or by ion exchange chromatography using an ionexchange column, e.g., a Sepharose™ ion exchange column available fromAmersham Biosciences. Either approach can be used to separatePEG-biomolecule isomers having the same molecular weight (positionalisomers).

Depending upon the intended use for the resulting PEG-conjugates,following conjugation, and optionally additional separation steps, theconjugate mixture may be concentrated, sterile filtered, and stored atlow temperatures from about −20° C. to about −80° C. Alternatively, theconjugate may be lyophilized, either with or without residual buffer andstored as a lyophilized powder. In some instances, it is preferable toexchange a buffer used for conjugation, such as sodium acetate, for avolatile buffer such as ammonium carbonate or ammonium acetate, that canbe readily removed during lyophilization, so that the lyophilizedprotein conjugate powder formulation is absent residual buffer.Alternatively, a buffer exchange step may be used using a formulationbuffer, so that the lyophilized conjugate is in a form suitable forreconstitution into a formulation buffer and ultimately foradministration to a mammal.

Precursor Maleimide Polymer Derivatives and Polymer Succinamic AcidConjugates

Precursor maleimide polymer derivatives useful in the present inventiongenerally comprise at least one maleimide substituent coupled to a watersoluble polymer segment. The maleimide substituent(s) can either becovalently bonded directly to a water soluble polymer segment, oralternatively can be connected to the polymer segment via a linkinggroup, L. A generalized structure is provided as I below, where theoptional linker is designated L, where L₀ indicates the absence of alinker, and L₁ indicates the presence of a linker.

The corresponding polymer maleamic acid, II, and polymer succinamic acidconjugates, IV and V, have structures as provided below. Since thestructures are all interrelated, the descriptions and embodimentsprovided herein for POLY and L apply equally to all of these structures.

The Polymer Segment

As shown in the illustrative structures above, the polymer reagents andconjugates of the invention contain a water-soluble polymer segment.Representative POLYs include poly(alkylene glycols) such aspoly(ethylene glycol), poly(propylene glycol) (“PPG”), copolymers ofethylene glycol and propylene glycol, poly(olefinic alcohol),poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide),poly(hydroxyalkylmethacrylate), poly(saccharides), poly(α-hydroxy acid),poly(vinyl alcohol), polyphosphazene, polyoxazoline, andpoly(N-acryloylmorpholine). POLY can be a homopolymer, an alternatingcopolymer, a random copolymer, a block copolymer, an alternatingtripolymer, a random tripolymer, or a block tripolymer of any of theabove. The water-soluble polymer segment is preferably, although notnecessarily, a polyethylene glycol, “PEG”, or a derivative thereof.

The polymer segment can have any of a number of different geometries,for example, POLY can be linear, branched, or forked. Most typically,POLY is linear or is branched, for example, having 2 polymer arms.Although much of the discussion herein is focused upon PEG as anillustrative POLY, the discussion and structures presented herein can bereadily extended to encompass any of the water-soluble polymer segmentsdescribed above.

Any water-soluble polymer having at least one reactive maleimideterminus can be used to prepare a polymer succinamic acid conjugate inaccordance with the invention and the invention is not limited in thisregard. Although water-soluble polymers bearing only a single reactivemaleimide can be used, polymers bearing two, three, four, five, six,seven, eight, nine, ten, eleven, twelve or more reactive maleimidessuitable for conversion to their open ring forms as set forth herein canbe used. Nonlimiting examples of the upper limit of the number ofmaleimide or amino precursor moieties associated with the water-solublepolymer segment include from about 1 to about 500, from 1 to about 100,from about 1 to about 80, from about 1 to about 40, from about 1 toabout 20, and from about 1 to about 10.

In turning now to the preferred POLY, PEG encompasses poly(ethyleneglycol) in any of its linear, branched or multi-arm forms, includingend-capped PEG, forked PEG, branched PEG, pendant PEG, and lesspreferably, PEG containing one or more degradable linkage separating themonomer subunits, to be more fully described below.

A PEG polymer segment comprises the following: —(CH₂CH₂O)_(n)—CH₂CH₂—,where (n) typically ranges from about 3 to about 4,000, or from about 3to about 3,000, or more preferably from about 20 to about 1,000.

POLY can be end-capped, for example an end-capped PEG where PEG isterminally capped with an inert end-capping group. Preferred end-cappedPEGs are those having as an end-capping moiety such as alkoxy,substituted alkoxy, alkenyloxy, substituted alkenyloxy, alkynyloxy,substituted alkynyloxy, aryloxy, substituted aryloxy. Preferredend-capping groups are methoxy, ethoxy, and benzyloxy. The end-cappinggroup can also advantageously comprise a phospholipid. Exemplaryphospholipids include phosphatidylcholines, such asdilauroylphosphatidylcholine, dioleylphosphatidylcholine,dipalmitoylphosphatidylcholine, disteroylphosphatidylcholine,behenoylphosphatidylcholine, arachidoylphosphatidylcholine, andlecithin. In one embodiment, however, a polymer of the invention issubstantially absent fatty acid groups or other lipophilic moieties.

Referring now to any of the structures containing a polymer segment,POLY, POLY may correspond or comprise the following:

“Z—(CH₂CH₂O)_(n)—” or “Z—(CH₂CH₂O)_(n)—CH₂CH₂—”,

where n ranges from about 3 to about 4000, or from about 10 to about4000, and Z is or includes a functional group, which may be a reactivegroup or an end-capping group. Examples of Z include hydroxy, amino,ester, carbonate, aldehyde, acetal, aldehyde hydrate, ketone, ketal,ketone hydrate, alkenyl, acrylate, methacrylate, acrylamide, sulfone,thiol, carboxylic acid, isocyanate, isothiocyanate, hydrazide, urea,maleimide, vinylsulfone, dithiopyridine, vinylpyridine, iodoacetamide,alkoxy, benzyloxy, silane, lipid, phospholipid, biotin, and fluorescein,including activated and protected forms thereof where applicable.Preferred are functional groups such as N-hydroxysuccinimidyl ester,benzotriazolyl carbonate, amine, vinylsulfone, maleimide, N-succinimidylcarbonate, hydrazide, succinimidyl propionate, succinimidyl butanoate,succinimidyl succinate, succinimidyl ester, glycidyl ether,oxycarbonylimidazole, p-nitrophenyl carbonate, aldehyde,orthopyridyl-disulfide, and acrylol.

These and other functional groups, Z, are described in the followingreferences, all of which are incorporated by reference herein:N-succinimidyl carbonate (see e.g., U.S. Pat. Nos. 5,281,698,5,468,478), amine (see, e.g., Buckmann et al. Makromol. Chem. 182:1379(1981), Zalipsky et al. Eur. Polym. J. 19:1177 (1983)), hydrazide (See,e.g., Andresz et al. Makromol. Chem. 179:301 (1978)), succinimidylpropionate and succinimidyl butanoate (see, e.g., Olson et al. inPoly(ethylene glycol) Chemistry & Biological Applications, pp 170-181,Harris & Zalipsky Eds., ACS, Washington, D.C., 1997; see also U.S. Pat.No. 5,672,662), succinimidyl succinate (See, e.g., Abuchowski et al.Cancer Biochem. Biophys. 7:175 (1984) and Joppich et al., Makromol.Chem. 180:1381 (1979), succinimidyl ester (see, e.g., U.S. Pat. No.4,670,417), benzotriazole carbonate (see, e.g., U.S. Pat. No.5,650,234), glycidyl ether (see, e.g., Pitha et al. Eur. J. Biochem.94:11 (1979), Elling et al., Biotech. Appl. Biochem. 13:354 (1991),oxycarbonylimidazole (see, e.g., Beauchamp, et al., Anal. Biochem.131:25 (1983), Tondelli et al. J. Controlled Release 1:251 (1985)),p-nitrophenyl carbonate (see, e.g., Veronese, et al., Appl. Biochem.Biotech., 11:141 (1985); and Sartore et al., Appl. Biochem. Biotech.,27:45 (1991)), aldehyde (see, e.g., Harris et al. J. Polym. Sci. Chem.Ed. 22:341 (1984), U.S. Pat. No. 5,824,784, U.S. Pat. No. 5,252,714),maleimide (see, e.g., Goodson et al. Bio/Technology 8:343 (1990), Romaniet al. in Chemistry of Peptides and Proteins 2:29 (1984)), and Kogan,Synthetic Comm. 22:2417 (1992)), orthopyridyl-disulfide (see, e.g.,Woghiren, et al. Bioconj. Chem. 4:314 (1993)), acrylol (see, e.g.,Sawhney et al., Macromolecules, 26:581 (1993)), vinylsulfone (see, e.g.,U.S. Pat. No. 5,900,461).

Again, the POLY structures shown immediately above may represent linearpolymer segments, or may form part of a branched or forked polymersegment. In an instance where the polymer segment is branched, the POLYstructures immediately above may, for example, correspond to the polymerarms forming part of the overall POLY structure. Alternatively, in aninstance where POLY possesses a forked structure, the above POLYstructure may, for example, correspond to the linear portion of thepolymer segment prior to the branch point.

POLY may also correspond to a branched PEG molecule having 2 arms, 3arms, 4 arms, 5 arms, 6 arms, 7 arms, 8 arms or more. Branched polymersused to prepare the polymer maleimides of the invention may possessanywhere from 2 to 300 or so reactive termini. Preferred are branchedpolymer segments having 2 or 3 polymer arms. An illustrative branchedPOLY, as described in U.S. Pat. No. 5,932,462, corresponds to thestructure:

In this representation, R″ is a nonreactive moiety, such as H, methyl ora PEG, and P and Q are nonreactive linkages. In a preferred embodiment,the branched PEG polymer segment is methoxy poly(ethylene glycol)disubstituted lysine, and corresponds to:

In the above particular branched configuration, the branched polymersegment possesses a single reactive site extending from the “C” branchpoint for positioning of the reactive maleimide group via a linker asdescribed herein. Branched PEGs such as these for use in the presentinvention will typically have fewer than 4 PEG arms, and morepreferably, will have 2 or 3 PEG arms. Such branched PEGs offer theadvantage of having a single reactive site, coupled with a larger, moredense polymer cloud than their linear PEG counterparts.

One particular type of branched PEG maleimide corresponds to thestructure: (MeO-PEG-)_(i)G-L_(0,1)-MAL, where MAL represents maleimide,i equals 2 or 3, and G is a lysine or other suitable amino acid residue.

An illustrative branched polymer maleimide of the invention has thestructure shown below, where L is any of the herein described linkers.

An illustrative PEG maleimide having a branched structure as showngenerally above corresponds to structure VII.

Branched PEGs for use in preparing a polymer maleimide of the inventionadditionally include those represented more generally by the formulaR(PEG)_(n), where R is a central or core molecule from which extends 2or more PEG arms. The variable n represents the number of PEG arms,where each of the polymer arms can independently be end-capped oralternatively, possess a reactive functional group at its terminus, suchas a maleimide or other reactive functional group. In such multi-armedembodiments of the invention, each PEG arm typically possesses amaleimide group at its terminus. Branched PEGs such as those representedgenerally by the formula, R(PEG)_(d), above possess 2 polymer arms toabout 300 polymer arms (i.e., n ranges from 2 to about 300). BranchedPEGs such as these preferably possess from 2 to about 25 polymer arms,more preferably from 2 to about 20 polymer arms, and even morepreferably from 2 to about 15 polymer arms or fewer. Most preferred aremulti-armed polymers having 3, 4, 5, 6, 7 or 8 arms.

Preferred core molecules in branched PEGs as described above arepolyols. Such polyols include aliphatic polyols having from 1 to 10carbon atoms and from 1 to 10 hydroxyl groups, including ethyleneglycol, alkane diols, alkyl glycols, alkylidene alkyl diols, alkylcycloalkane diols, 1,5-decalindiol,4,8-bis(hydroxymethyl)tricyclodecane, cycloalkylidene diols,dihydroxyalkanes, trihydroxyalkanes, and the like. Cycloaliphaticpolyols may also be employed, including straight chained or closed-ringsugars and sugar alcohols, such as mannitol, sorbitol, inositol,xylitol, quebrachitol, threitol, arabitol, erythritol, adonitol,dulcitol, facose, ribose, arabinose, xylose, lyxose, rhamnose,galactose, glucose, fructose, sorbose, mannose, pyranose, altrose,talose, tagitose, pyranosides, sucrose, lactose, maltose, and the like.Additional aliphatic polyols include derivatives of glyceraldehyde,glucose, ribose, mannose, galactose, and related stereoisomers. Othercore polyols that may be used include crown ether, cyclodextrins,dextrins and other carbohydrates such as starches and amylose. Preferredpolyols include glycerol, pentaerythritol, sorbitol, andtrimethylolpropane.

A representative multi-arm polymer structure of the type described aboveis:

where d is an integer from 3 to about 100, and R is a residue of acentral core molecule having 3 or more hydroxyl groups, amino groups, orcombinations thereof.

Multi-armed PEGs for use in preparing a polymer maleimide of theinvention include multi-arm PEGs available from Nektar, Huntsville, Ala.In a preferred embodiment, a multi-armed polymer maleimide of theinvention corresponds to the following, where the specifics of thelinkered maleimide portion of the molecule are provided elsewhereherein.

where

PEG is —(CH₂CH₂O)_(n)CH₂CH₂—,

M is:

and m is selected from the group consisting of 3, 4, 5, 6, 7, and 8.

Alternatively, the polymer maleimide may possess an overall forkedstructure. An example of a forked PEG corresponds to the structure:

where PEG is any of the forms of PEG described herein, A is a linkinggroup, preferably a hydrolytically stable linkage such as oxygen,sulfur, or —C(O)—NH—, F and F′ are hydrolytically stable spacer groupsthat are optionally present, and the other variables, L and maleimide(MAL) are as defined above. Exemplary linkers and spacer groupscorresponding to A, F and F′ are described in International ApplicationNo. PCT/US99/05333, and are useful in forming polymer segments of thistype for use in the present invention. F and F′ are spacer groups thatmay be the same of different. In one particular embodiment of the above,PEG is mPEG, A corresponds to —C(O)—NH—, and F and F′ are both methyleneor —CH₂—. This type of polymer segment is useful for reaction with twoactive agents, where the two active agents are positioned a precise orpredetermined distance apart, depending upon the selection of F and F'.

An illustrative branched, forked PEG has the structure shown below,where the branched portion is on the left, and the forked portion havingtwo maleimide groups extending therefrom in on the right.

Alternatively, the PEG polymer segment for use in preparing a polymermaleimide of the invention may be a PEG molecule having pendant reactivegroups along the length of the PEG chain rather than at the end(s), toyield a stabilized polymer maleimide having one or more pendantmaleimide groups attached to the PEG chain by a linker, L.

Further, in a less preferred embodiment, the polymer segment itself maypossess one or more weak or degradable linkages that are subject tohydrolysis. Illustrative degradable linkages that may be present in thepolymer segment include but are not limited to carbonate, imine,phosphate ester, and hydrazone.

Generally, the nominal average molecular mass of the water-solublepolymer segment, POLY will vary. The nominal average molecular mass ofPOLY typically falls in one or more of the following ranges: about 100daltons to about 100,000 daltons; from about 500 daltons to about 80,000daltons; from about 1,000 daltons to about 50,000 daltons; from about2,000 daltons to about 25,000 daltons; from about 5,000 daltons to about20,000 daltons. Exemplary nominal average molecular masses for thewater-soluble polymer segment POLY include about 1,000 daltons, about5,000 daltons, about 10,000 daltons, about 15,000 daltons, about 20,000daltons, about 25,000 daltons, about 30,000 daltons, and about 40,000daltons. Low molecular weight POLYs possess molecular masses of about250, 500, 750, 1000, 2000, or 5000 daltons.

Any of the above structures corresponding to a polymer-maleimide ismeant to also encompass its corresponding polymer succinamic acidcounterpart, even if not explicitly shown. Thus, all polymer maleimidestructures herein are meant to extend to the same structure with theexception that the maleimide ring is in its open-ring form, and can beunconjugated (maleamic acid) or conjugated (succinamic acid conjugate).

The Linker

In turning now to the linker moiety, a linker moiety or simply “linker”of the invention is represented generally by the variable, L. A linkerof the invention, L, if present, typically contains from about 1 toabout 40 atoms. The linker is the portion of the overall polymer thatlinks the maleimide or maleamic acid or succinamic acid portion of thepolymer with the polymer segment. A linker of the invention may be asingle atom, such as an oxygen or a sulfur, two atoms, or a number ofatoms. A linker is typically but is not necessarily linear in nature.The overall length of the linker will typically range between 1 to about40 atoms, where by length is meant the number of atoms in a singlechain, not counting substituents. For instance, —CH₂— counts as one atomwith respect to overall linker length, —CH₂CH₂O— counts as 3 atoms inlength. Preferably, a linker will have a length of about 1 to about 20atoms, or from about 2 to about 15 atoms, or from about 1 to about 6atoms, and is hydrolytically stable.

A linker of the invention can be a single functional group such as anamide, an ester, a urethane, or a urea, or may contain methylene orother alkylene groups flanking either side of the single functionalgroup. Alternatively, a linker may contain a combination of functionalgroups that can be the same or different. Additionally, a linker of theinvention can be an alkylene chain, optionally containing one or moreoxygen or sulfur atoms (i.e., an ether or thioether). Preferred linkersare those that are hydrolytically stable. When viewed in the context ofthe structures herein, a linker is one that when considered as part ofthe overall polymer, does not result in an overall structure containinga peroxide bond (—O—O—) or an —N—O— or —O—N— bond.

In the context of structures I, II a linker of the invention may be anyof the following: —O—, —NH—, —S—, —C(O)—, C(O)—NH, NH—C(O)—NH,O—C(O)—NH, —C(S)—, —CH₂—, —CH₂—CH₂—, —CH₂—CH₂—CH₂—, —CH₂—CH₂—CH₂—CH₂—,—O—CH₂—, —CH₂—O—, —O—CH₂—CH₂—, —CH₂—O—CH₂—, —CH₂—CH₂—O—,—O—CH₂—CH₂—CH₂—, —CH₂—O—CH₂—CH₂—, —CH₂—CH₂—O—CH₂—, —CH₂—CH₂—CH₂—O—,—O—CH₂—CH₂—CH₂—CH₂—, —CH₂—O—CH₂—CH₂—CH₂—, —CH₂—CH₂—O—CH₂—CH₂—,—CH₂—CH₂—CH₂—O—CH₂—, —CH₂—CH₂—CH₂—CH₂—O—, —C(O)—NH—CH₂—,—C(O)—NH—CH₂—CH₂—, —CH₂—C(O)—NH—CH₂—, —CH₂—CH₂—C(O)—NH—,—C(O)—NH—CH₂—CH₂—CH₂—, —CH₂—C(O)—NH—CH₂—CH₂—, —CH₂—CH₂—C(O)—NH—CH₂—,—CH₂—CH₂—CH₂—C(O)—NH—, —C(O)—NH—CH₂—CH₂—CH₂—CH₂—,—CH₂—C(O)—NH—CH₂—CH₂—CH₂—, —CH₂—CH₂—C(O)—NH—CH₂—CH₂—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—,—CH₂—CH₂—CH₂—CH₂—C(O)—NH—, —C(O)—O—CH₂—, —CH₂—C(O)—O—CH₂—,—CH₂—CH₂—C(O)—O—CH₂—, —C(O)—O—CH₂—CH₂—, —NH—C(O)—CH₂—,—CH₂—NH—C(O)—CH₂—, —CH₂—CH₂—NH—C(O)—CH₂—, —NH—C(O)—CH₂—CH₂—,—CH₂—NH—C(O)—CH₂—CH₂, —CH₂—CH₂—NH—C(O)—CH₂—CH₂, —C(O)—NH—CH₂—,—C(O)—NH—CH₂—CH₂—, —O—C(O)—NH—CH₂—, —O—C(O)—NH—CH₂—CH₂—, —NH—CH₂—,—NH—CH₂—CH₂—, —CH₂—NH—CH₂—, —CH₂—CH₂—NH—CH₂—, —C(O)—CH₂—,—C(O)—CH₂—CH₂—, —CH₂—C(O)—CH₂—, —CH₂—CH₂—C(O)—CH₂—,—CH₂—CH₂—C(O)—CH₂—CH₂—, —CH₂—CH₂—C(O)—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—CH₂—, bivalent cycloalkylene group,—N(R⁶)—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—CH₂—CH₂—,—O—C(O)—NH—[CH₂]_(h)—(OCH₂CH₂)_(j)—, and combinations of two or more ofany of the foregoing, wherein (h) is 0 to 6, (j) is 0 to 20, R⁶ is H oran organic radical selected from the group consisting of alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl and substituted aryl.

For purposes of the present disclosure, however, a series of atoms isnot considered as a linker moiety when the series of atoms isimmediately adjacent to a polymer segment, POLY, and the series of atomsis but another monomer such that the proposed linker moiety wouldrepresent a mere extension of the polymer chain. For example, given thepartial structure “POLY-L-,” where POLY in this instance is defined as“CH₃—O—(CH₂CH₂O)_(n)—”, the linker moiety would not be “—CH₂CH₂O—” sincesuch a definition would merely represent an extension of the polymer.That is not to say, however, that a linker of the invention cannotpossess one or more contiguous —CH₂CH₂O— portions. For example, a linkermay contain one or more (—CH₂CH₂O—) subunits flanked on one or bothsides by one or a combination of illustrative linkers as provided above.

In one embodiment of the invention, a linker possesses the structure:

In the above linker, R¹ and R² in each occurrence are each independentlyH or an organic radical that is selected from the group consisting ofalkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl,alkylenecycloalkyl, and substituted alkylenecycloalkyl. In the abovestructure, a subscript of zero indicates the absence of that particularatom or functional group.

Using a single maleimide end group and a methoxy cap as arepresentation, certain exemplary PEG maleimide structures areillustrated in Structures 1-4 below. The linkers, L, shown in Table 1,may be used to form the maleamic acid polymers and conjugates of theinvention. The PEG maleimide represented by Structure 3-ET is called“linkerless” since the maleimide ring simply replaces the terminalhydroxyl group in the PEG. The exemplary linkers shown below can beutilized in combination with any of the above described polymersegments; the embodiments below with mPEG are meant only to beillustrative.

TABLE 1

Linker Abbrev. X AMET —(CH₂)₂— AMTR —(CH₂)₃— AMPE —(CH₂)₅— MCH

TEO

mPH

pPHAL

L₂ = —NH—Y— Linker Abbrev. Y BU —(CH₂)₄— HE —(CH₂)₆— L₂ = —O—Z— LinkerAbbrev. Z ET —(CH₂)₂— PR —(CH₂)₃— PRAC —C(O)—CH₂CH₂— L₂ = —CH₂—Q LinkerAbbrev. Q PACA —C(O)— PAME —C(O)—NH—CH₂— PAET —C(O)—NH—CH₂CH₂— BAET—(CH₂)—C(O)NH—CH₂CH₂— PAHE —C(O)—NH—(CH₂)₆— BAET —CH₂—C(O)NH—(CH₂)₆—PAOX —C(O)—NH—CH₂CH₂O—

Generally, preferred are linkers that are effective to provide a rate ofring opening hydrolysis of the uncoupled polymer maleimide that isincreased (i.e., faster) than that of the same water soluble polymermaleimide absent a linker. In a preferred embodiment, the linking groupfacilitates ring opening such that the ring opening hydrolysis rate ofthe maleimide has a half life equal to or shorter than about 12 hours atpH 7.5 when measured at room temperature. In a more preferredembodiment, the linking group facilitates ring opening such that thering opening hydrolysis rate of the maleimide has a half life equal toor shorter than about 12 hours at pH 9 when measured at roomtemperature. Preferred linking groups that facilitate ring openinginclude the linkerless maleimides, i.e. L₃-ET, those with short alkyllinkers, e.g. L₃-PR, those with an ethylene or aryl group attached tothe maleimide ring nitrogen, e.g. L₁-MCH and L₁-pPHAL, those with shortalkyl linkers between the maleimide nitrogen atom and a carbonyl group,e.g. L₁-AMET and various modifications of the listed groups that containsubstituents that enhance electron withdrawal from the maleimide ringnitrogen without providing significant steric hindrance to hydrolysis,i.e. no branching substitution to the linker atom attached to the ringnitrogen. Preferred linkers possess an electron withdrawing group withinabout 6 atoms of the maleimide or maleimide-derived nitrogen, i.e.,within 1, 2, 3, 4, 5, or 6 atoms of the maleimide or maleimide derivednitrogen, or even more preferably, within about 3 atoms.

Polymers and conjugates of the invention include monofunctional,bifunctional, and multi-functional structures as previously described.

For instance, a polymer maleimide precursor of a polymer or conjugate ofthe invention may be described generally by the following structurewhere the variables are as defined elsewhere herein:

In the above embodiment, the L's may be the same of different. In oneparticular embodiment the polymer reagent is homo-bifunctional, that isto say, both L's are the same.

Succinamic Acid Conjugates

The generalized features of the conjugates of the invention have beendescribed in detailed fashion above. Active agents that are covalentlyattached to a polymer succinamic acid encompass any of a number of typesof molecules, entities, surfaces, and the like, as will become apparentfrom the following.

Target Molecules and Surfaces

The polymer maleimides (both open and closed ring) of the invention maybe attached, either covalently or non-covalently, to a number ofentities including films, chemical separation and purification surfaces,solid supports, metal/metal oxide surfaces such as gold, titanium,tantalum, niobium, aluminum, steel, and their oxides, silicon oxide,macromolecules, and small molecules. Additionally, the polymers andmethods of the invention may also be used in biochemical sensors,bioelectronic switches, and gates. The polymers and methods of theinvention may also be employed in preparing carriers for peptidesynthesis, for the preparation of polymer-coated surfaces and polymergrafts, to prepare polymer-ligand conjugates for affinity partitioning,to prepare cross-linked or non-cross-linked hydrogels, and to preparepolymer-cofactor adducts for bioreactors.

A biologically active agent for use in providing a conjugate of theinvention may be any one or more of the following. Suitable agents maybe selected from, for example, hypnotics and sedatives, psychicenergizers, tranquilizers, respiratory drugs, anticonvulsants, musclerelaxants, antiparkinson agents (dopamine antagnonists), analgesics,anti-inflammatories, antianxiety drugs (anxiolytics), appetitesuppressants, antimigraine agents, muscle contractants, anti-infectives(antibiotics, antivirals, antifungals, vaccines) antiarthritics,antimalarials, antiemetics, anepileptics, bronchodilators, cytokines,growth factors, anti-cancer agents, antithrombotic agents,antihypertensives, cardiovascular drugs, antiarrhythmics, antioxicants,anti-asthma agents, hormonal agents including contraceptives,sympathomimetics, diuretics, lipid regulating agents, antiandrogenicagents, antiparasitics, anticoagulants, neoplastics, antineoplastics,hypoglycemics, nutritional agents and supplements, growth supplements,antienteritis agents, vaccines, antibodies, diagnostic agents, andcontrasting agents.

More particularly, the active agent may fall into one of a number ofstructural classes, including but not limited to small molecules(preferably insoluble small molecules), peptides, polypeptides,proteins, antibodies, polysaccharides, steroids, nucleotides,oligonucleotides, polynucleotides, fats, electrolytes, and the like.Preferably, an active agent for coupling to a polymer maleimidepossesses a native amino or a sulfydryl group, or alternatively, ismodified to contain at least one reactive amino or sulfhydryl groupsuitable for coupling to a polymer maleimide.

Specific examples of active agents suitable for covalent attachment to apolymer of the invention include but are not limited to aspariginase,amdoxovir (DAPD), antide, becaplermin, calcitonins, cyanovirin,denileukin diftitox, erythropoietin (EPO), EPO agonists (e.g., peptidesfrom about 10-40 amino acids in length and comprising a particular coresequence as described in WO 96/40749), dornase alpha, erythropoiesisstimulating protein (NESP), coagulation factors such as Factor V, FactorVII, Factor VIIa, Factor VIII, Factor IX, Factor X, Factor XII, FactorXIII, von Willebrand factor; ceredase, cerezyme, alpha-glucosidase,collagen, cyclosporin, alpha defensins, beta defensins, exedin-4,granulocyte colony stimulating factor (GCSF), thrombopoietin (TPO),alpha-1 proteinase inhibitor, elcatonin, granulocyte macrophage colonystimulating factor (GMCSF), fibrinogen, filgrastim, growth hormoneshuman growth hormone (hGH), growth hormone releasing hormone (GHRH),GRO-beta, GRO-beta antibody, bone morphogenic proteins such as bonemorphogenic protein-2, bone morphogenic protein-6, OP-1; acidicfibroblast growth factor, basic fibroblast growth factor, CD-40 ligand,heparin, human serum albumin, low molecular weight heparin (LMWH),interferons such as interferon alpha, interferon beta, interferon gamma,interferon omega, interferon tau, consensus interferon; interleukins andinterleukin receptors such as interleukin-1 receptor, interleukin-2,interleukin-2 fusion proteins, interleukin-1 receptor antagonist,interleukin-3, interleukin-4, interleukin-4 receptor, interleukin-6,interleukin-8, interleukin-12, interleukin-13 receptor, interleukin-17receptor; lactoferrin and lactoferrin fragments, luteinizing hormonereleasing hormone (LHRH), insulin, pro-insulin, insulin analogues (e.g.,mono-acylated insulin as described in U.S. Pat. No. 5,922,675), amylin,C-peptide, somatostatin, somatostatin analogs including octreotide,vasopressin, follicle stimulating hormone (FSH), influenza vaccine,insulin-like growth factor (IGF), insulintropin, macrophage colonystimulating factor (M-CSF), plasminogen activators such as alteplase,urokinase, reteplase, streptokinase, pamiteplase, lanoteplase, andteneteplase; nerve growth factor (NGF), osteoprotegerin,platelet-derived growth factor, tissue growth factors, transforminggrowth factor-1, vascular endothelial growth factor, leukemia inhibitingfactor, keratinocyte growth factor (KGF), glial growth factor (GGF), TCell receptors, CD molecules/antigens, tumor necrosis factor (TNF),monocyte chemoattractant protein-1, endothelial growth factors,parathyroid hormone (PTH), glucagon-like peptide, somatotropin, thymosinalpha 1, thymosin alpha 1 IIb/IIIa inhibitor, thymosin beta 10, thymosinbeta 9, thymosin beta 4, alpha-1 antitrypsin, phosphodiesterase (PDE)compounds, VLA-4 (very late antigen-4), VLA-4 inhibitors,bisphosphonates, respiratory syncytial virus antibody, cystic fibrosistransmembrane regulator (CFTR) gene, deoxyribonuclease (Dnase),bactericidal/permeability increasing protein (BPI), and anti-CMVantibody. Exemplary monoclonal antibodies include etanercept (a dimericfusion protein consisting of the extracellular ligand-binding portion ofthe human 75 kD TNF receptor linked to the Fc portion of IgG1),abciximab, afeliomomab, basiliximab, daclizumab, infliximab, ibritumomabtiuexetan, mitumomab, muromonab-CD3, iodine 131 tositumomab conjugate,olizumab, rituximab, and trastuzumab (herceptin).

Additional agents suitable for covalent attachment to a polymer includebut are not limited to amifostine, amiodarone, aminocaproic acid,aminohippurate sodium, aminoglutethimide, aminolevulinic acid,aminosalicylic acid, amsacrine, anagrelide, anastrozole, asparaginase,anthracyclines, bexarotene, bicalutamide, bleomycin, buserelin,busulfan, cabergoline, capecitabine, carboplatin, carmustine,chlorambucin, cilastatin sodium, cisplatin, cladribine, clodronate,cyclophosphamide, cyproterone, cytarabine, camptothecins, 13-cisretinoic acid, all trans retinoic acid; dacarbazine, dactinomycin,daunorubicin, deferoxamine, dexamethasone, diclofenac,diethylstilbestrol, docetaxel, doxorubicin, epirubicin, estramustine,etoposide, exemestane, fexofenadine, fludarabine, fludrocortisone,fluorouracil, fluoxymesterone, flutamide, gemcitabine, epinephrine,L-Dopa, hydroxyurea, idarubicin, ifosfamide, imatinib, irinotecan,itraconazole, goserelin, letrozole, leucovorin, levamisole, lisinopril,lovothyroxine sodium, lomustine, mechlorethamine, medroxyprogesterone,megestrol, melphalan, mercaptopurine, metaraminol bitartrate,methotrexate, metoclopramide, mexiletine, mitomycin, mitotane,mitoxantrone, naloxone, nicotine, nilutamide, octreotide, oxaliplatin,pamidronate, pentostatin, pilcamycin, porfimer, prednisone,procarbazine, prochlorperazine, ondansetron, raltitrexed, sirolimus,streptozocin, tacrolimus, tamoxifen, temozolomide, teniposide,testosterone, tetrahydrocannabinol, thalidomide, thioguanine, thiotepa,topotecan, tretinoin, valrubicin, vinblastine, vincristine, vindesine,vinorelbine, dolasetron, granisetron; formoterol, fluticasone,leuprolide, midazolam, alprazolam, amphotericin B, podophylotoxins,nucleoside antivirals, aroyl hydrazones, sumatriptan; macrolides such aserythromycin, oleandomycin, troleandomycin, roxithromycin,clarithromycin, davercin, azithromycin, flurithromycin, dirithromycin,josamycin, spiromycin, midecamycin, leucomycin, miocamycin, rokitamycin,and azithromycin, and swinolide A; fluoroquinolones such asciprofloxacin, ofloxacin, levofloxacin, trovafloxacin, alatrofloxacin,moxifloxicin, norfloxacin, enoxacin, grepafloxacin, gatifloxacin,lomefloxacin, sparfloxacin, temafloxacin, pefloxacin, amifloxacin,fleroxacin, tosufloxacin, prulifloxacin, irloxacin, pazufloxacin,clinafloxacin, and sitafloxacin; aminoglycosides such as gentamicin,netilmicin, paramecin, tobramycin, amikacin, kanamycin, neomycin, andstreptomycin, vancomycin, teicoplanin, rampolanin, mideplanin, colistin,daptomycin, gramicidin, colistimethate; polymixins such as polymixin B,capreomycin, bacitracin, penems; penicillins includingpenicllinase-sensitive agents like penicillin G, penicillin V;penicllinase-resistant agents like methicillin, oxacillin, cloxacillin,dicloxacillin, floxacillin, nafcillin; gram negative microorganismactive agents like ampicillin, amoxicillin, and hetacillin, cillin, andgalampicillin; antipseudomonal penicillins like carbenicillin,ticarcillin, azlocillin, mezlocillin, and piperacillin; cephalosporinslike cefpodoxime, cefprozil, ceftbuten, ceftizoxime, ceftriaxone,cephalothin, cephapirin, cephalexin, cephradrine, cefoxitin,cefamandole, cefazolin, cephaloridine, cefaclor, cefadroxil,cephaloglycin, cefuroxime, ceforanide, cefotaxime, cefatrizine,cephacetrile, cefepime, cefixime, cefonicid, cefoperazone, cefotetan,cefmetazole, ceftazidime, loracarbef, and moxalactam, monobactams likeaztreonam; and carbapenems such as imipenem, meropenem, pentamidineisethiouate, albuterol sulfate, lidocaine, metaproterenol sulfate,beclomethasone diprepionate, triamcinolone acetamide, budesonideacetonide, fluticasone, ipratropium bromide, flunisolide, cromolynsodium, and ergotamine tartrate; taxanes such as paclitaxel; SN-38, andtyrphostines.

Preferred peptides or proteins for coupling to a polymer maleimide ofthe invention include EPO, IFN-α, IFN-γ, consensus IFN, Factor VII,Factor VIII, Factor IX, IL-2, remicade (infliximab), Rituxan(rituximab), Enbrel (etanercept), Synagis (palivizumab), Reopro(abciximab), Herceptin (trastuzimab), tPA, Cerizyme (imiglucerase),Hepatitus-B vaccine, rDNAse, alpha-1 proteinase inhibitor, GCSF, GMCSF,hGH, insulin, FSH, and PTH.

The above exemplary biologically active agents are meant to encompass,where applicable, analogues, agonists, antagonists, inhibitors, isomers,and pharmaceutically acceptable salt forms thereof. In reference topeptides and proteins, the invention is intended to encompass synthetic,recombinant, native, glycosylated, and non-glycosylated forms, as wellas biologically active fragments thereof. The above biologically activeproteins are additionally meant to encompass variants having one or moreamino acids substituted (e.g., cysteine), deleted, or the like, as longas the resulting variant protein possesses at least a certain degree ofactivity of the parent (native) protein.

The conjugates or methods described herein can also be extended tohydrogel formulations.

Pharmaceutical Compositions

The present invention also includes pharmaceutical preparationscomprising a conjugate as provided herein in combination with apharmaceutical excipient. Generally, the conjugate itself will be in asolid form (e.g., a precipitate) or in solution, which can be combinedwith a suitable pharmaceutical excipient that can be in either solid orliquid form.

Exemplary excipients include, without limitation, those selected fromthe group consisting of carbohydrates, inorganic salts, antimicrobialagents, antioxidants, surfactants, buffers, acids, bases, andcombinations thereof.

A carbohydrate such as a sugar, a derivatized sugar such as an alditol,aldonic acid, an esterified sugar, and/or a sugar polymer may be presentas an excipient. Specific carbohydrate excipients include, for example:monosaccharides, such as fructose, maltose, galactose, glucose,D-mannose, sorbose, and the like; disaccharides, such as lactose,sucrose, trehalose, cellobiose, and the like; polysaccharides, such asraffinose, melezitose, maltodextrins, dextrans, starches, and the like;and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol,sorbitol (glucitol), pyranosyl sorbitol, myoinositol, and the like.

The excipient can also include an inorganic salt or buffer such ascitric acid, sodium chloride, potassium chloride, sodium sulfate,potassium nitrate, sodium phosphate monobasic, sodium phosphate dibasic,and combinations thereof.

The preparation may also include an antimicrobial agent for preventingor deterring microbial growth. Nonlimiting examples of antimicrobialagents suitable for the present invention include benzalkonium chloride,benzethonium chloride, benzyl alcohol, cetylpyridinium chloride,chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate,thimersol, and combinations thereof.

An antioxidant can be present in the preparation as well. Antioxidantsare used to prevent oxidation, thereby preventing the deterioration ofthe conjugate or other components of the preparation. Suitableantioxidants for use in the present invention include, for example,ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene,hypophosphorous acid, monothioglycerol, propyl gallate, sodiumbisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite, andcombinations thereof.

A surfactant may be present as an excipient. Exemplary surfactantsinclude: polysorbates, such as “Tween 20” and “Tween 80,” and pluronicssuch as F68 and F88 (both of which are available from BASF, Mount Olive,N.J.); sorbitan esters; lipids, such as phospholipids such as lecithinand other phosphatidylcholines, phosphatidylethanolamines (althoughpreferably not in liposomal form), fatty acids and fatty esters;steroids, such as cholesterol; and chelating agents, such as EDTA, zincand other such suitable cations.

Acids or bases may be present as an excipient in the preparation.Nonlimiting examples of acids that can be used include those acidsselected from the group consisting of hydrochloric acid, acetic acid,phosphoric acid, citric acid, malic acid, lactic acid, formic acid,trichloroacetic acid, nitric acid, perchloric acid, phosphoric acid,sulfuric acid, fumaric acid, and combinations thereof. Examples ofsuitable bases include, without limitation, bases selected from thegroup consisting of sodium hydroxide, sodium acetate, ammoniumhydroxide, potassium hydroxide, ammonium acetate, potassium acetate,sodium phosphate, potassium phosphate, sodium citrate, sodium formate,sodium sulfate, potassium sulfate, potassium fumerate, and combinationsthereof.

The pharmaceutical preparations encompass all types of formulations andin particular those that are suited for injection, e.g., powders thatcan be reconstituted as well as suspensions and solutions. The amount ofthe conjugate (i.e., the conjugate formed between the active agent andthe polymer described herein) in the composition will vary depending ona number of factors, but will optimally be a therapeutically effectivedose when the composition is stored in a unit dose container (e.g., avial). In addition, the pharmaceutical preparation can be housed in asyringe. A therapeutically effective dose can be determinedexperimentally by repeated administration of increasing amounts of theconjugate in order to determine which amount produces a clinicallydesired endpoint.

The amount of any individual excipient in the composition will varydepending on the activity of the excipient and particular needs of thecomposition. Typically, the optimal amount of any individual excipientis determined through routine experimentation, i.e., by preparingcompositions containing varying amounts of the excipient (ranging fromlow to high), examining the stability and other parameters, and thendetermining the range at which optimal performance is attained with nosignificant adverse effects.

Generally, however, the excipient will be present in the composition inan amount of about 1% to about 99% by weight, preferably from about5%-98% by weight, more preferably from about 15-95% by weight of theexcipient, with concentrations less than 30% by weight most preferred.

These foregoing pharmaceutical excipients along with other excipientsare described in “Remington: The Science & Practice of Pharmacy”,19^(th) ed., Williams & Williams, (1995), the “Physician's DeskReference”, 52^(nd) ed., Medical Economics, Montvale, N.J. (1998), andKibbe, A. H., Handbook of Pharmaceutical Excipients, 3^(rd) Edition,American Pharmaceutical Association, Washington, D.C., 2000.

The pharmaceutical preparations of the present invention are typically,although not necessarily, administered via injection and are thereforegenerally liquid solutions or suspensions immediately prior toadministration. The pharmaceutical preparation can also take other formssuch as syrups, creams, ointments, tablets, powders, and the like. Othermodes of administration are also included, such as pulmonary, rectal,transdermal, transmucosal, oral, intrathecal, subcutaneous,intra-arterial, and so forth.

As previously described, the conjugates can be administered injectedparenterally by intravenous injection, or less preferably byintramuscular or by subcutaneous injection. Suitable formulation typesfor parenteral administration include ready-for-injection solutions, drypowders for combination with a solvent prior to use, suspensions readyfor injection, dry insoluble compositions for combination with a vehicleprior to use, and emulsions and liquid concentrates for dilution priorto administration, among others.

Methods of Administering

The invention also provides a method for administering a conjugate asprovided herein to a patient suffering from a condition that isresponsive to treatment with conjugate. The method comprisesadministering, generally via injection, a therapeutically effectiveamount of the conjugate (preferably provided as part of a pharmaceuticalpreparation). The method of administering may be used to treat anycondition that can be remedied or prevented by administration of theparticular conjugate. Those of ordinary skill in the art appreciatewhich conditions a specific conjugate can effectively treat. The actualdose to be administered will vary depend upon the age, weight, andgeneral condition of the subject as well as the severity of thecondition being treated, the judgment of the health care professional,and conjugate being administered. Therapeutically effective amounts areknown to those skilled in the art and/or are described in the pertinentreference texts and literature. Generally, a therapeutically effectiveamount will range from about 0.001 mg to 100 mg, preferably in dosesfrom 0.01 mg/day to 75 mg/day, and more preferably in doses from 0.10mg/day to 50 mg/day.

The unit dosage of any given conjugate (again, preferably provided aspart of a pharmaceutical preparation) can be administered in a varietyof dosing schedules depending on the judgment of the clinician, needs ofthe patient, and so forth. The specific dosing schedule will be known bythose of ordinary skill in the art or can be determined experimentallyusing routine methods. Exemplary dosing schedules include, withoutlimitation, administration five times a day, four times a day, threetimes a day, twice daily, once daily, three times weekly, twice weekly,once weekly, twice monthly, once monthly, and any combination thereof.Once the clinical endpoint has been achieved, dosing of the compositionis halted.

One advantage of administering the conjugates of the present inventionis that individual water-soluble polymer portions can be cleaved off.Such a result is advantageous when clearance from the body ispotentially a problem because of the polymer size. Optimally, cleavageof each water-soluble polymer portion is facilitated through the use ofphysiologically cleavable and/or enzymatically degradable linkages suchas urethane, amide, carbonate or ester-containing linkages. In this way,clearance of the conjugate (via cleavage of individual water-solublepolymer portions) can be modulated by selecting the polymer molecularsize and the type functional group that would provide the desiredclearance properties. One of ordinary skill in the art can determine theproper molecular size of the polymer as well as the cleavable functionalgroup. For example, one of ordinary skill in the art, using routineexperimentation, can determine a proper molecular size and cleavablefunctional group by first preparing a variety of polymer derivativeswith different polymer weights and cleavable functional groups, and thenobtaining the clearance profile (e.g., through periodic blood or urinesampling) by administering the polymer derivative to a patient andtaking periodic blood and/or urine sampling. Once a series of clearanceprofiles have been obtained for each tested conjugate, a suitableconjugate can be identified.

All articles, books, patents, patent publications and other publicationsreferenced herein are incorporated by reference in their entireties.

EXAMPLES

It is to be understood that while the invention has been described inconjunction with certain preferred specific embodiments thereof, theforegoing description as well as the examples that follow are intendedto illustrate and not limit the scope of the invention. Other aspects,advantages and modifications within the scope of the invention will beapparent to those skilled in the art to which the invention pertains.

Abbreviations.

DCM: dichloromethane

NMR: nuclear magnetic resonance

DI: deionized

r.t. room temperature

anh. anhydrous

Da Daltons

GPC Gel Permeation Chromatography

Materials and Methods.

All chemical reagents referred to in the appended examples arecommercially available unless otherwise indicated.

All PEG reagents referred to in the appended examples are available fromNektar, Huntsville, Ala. All ¹HNMR data was generated by a 300 or 400MHz NMR spectrometer manufactured by Bruker.

Example 1 Hydrolysis Rates of Exemplary Linkered PEG Maleimides

A series of representative methoxy-PEG maleimides with an averagemolecular weight of 5000 Daltons was synthesized and studied. Thekinetics of the hydrolysis reaction of the maleimide ring for eachstructure below was determined by measuring the UV absorption at 297 nmof solutions of each mPEG maleimide at a concentration of 5 mg/mL in 50mM Phosphate Buffer at pH of approximately 7.5.

The generalized structure for the polymer maleimides is shown below.Exact structures corresponding to each of the linkers (L₁, L₂, and L₃)is provided in Table 1 above.

TABLE 2 Hydrolysis Rates of mPEG (5 k-Da) Maleimides (5 mg/mL) in 50 mMPhosphate Buffer (pH ~7.5) as Measured by UV Absorption at 297 nmStructure half-life (hrs) Relative Rate L₁-AMTR 8.8 3.66 L₁-AMPE 19.41.66 L₁-MCH 16.3 1.98 L₂-BU 19.6 1.65 L₂-HE 32.3 1.00 L₃-ET 8.1 4.01L₃-PR 11.5 2.82

As shown by the data in Table 2, the hydrolysis rates of theillustrative polymer maleimides vary with structure. In this group, theHE linker is the most resistant to hydrolysis, while the ET linkerexhibits the fastest hydrolysis rate, indicating the tendency of itsmaleimide ring towards hydrolysis, even at fairly mild pHs.

The data above indicates that preferred linking groups for facilitatingring opening include those having a strong electron-withdrawing group,EWG, in close proximity (most preferably within 3 or so atoms) to themaleimide substituent(s), i.e., the nitrogen of the maleimide ring. TheL₃-ET linker, —O-ethylene-, possesses an electron withdrawing atom,oxygen, within 3 atoms of the maleimide nitrogen, which appears tocontribute to its tendency towards an enhanced rate of hydrolysis.Preferred are linkers having an EWG most preferably within 1, 2, 3 or 4atoms of the maleimide nitrogen.

Example 2

Hydrolysis of a Branched and Tinkered polymer maleimide, mPEG2-MAL-40K

The polymer maleimide pictured above, mPEG2-MAL-40K, was obtained fromNektar (Huntsville, Ala.). This polymer derivative undergoes a limiteddegree of hydrolysis of the maleimide ring under certain conditions toform the corresponding maleamic acid derivative, as described below.

The hydrolysis reaction was monitored analytically by observing thepercentage decrease of the parent maleimide over time by HPLC. Thekinetics of the hydrolysis reaction was determined at a pH of about 5.5,using a HEPES buffered solution at approximately 25° C. A linearcorrelation was obtained from the raw data by plotting either thelogarithm of the concentration of either the maleamic acid or themaleimide versus time (the latter is shown in FIG. 3).

That data was then used to determine the half-life of the hydrolysisreaction, which was calculated to be approximately 34 days under theconditions examined. Thus, under these conditions, this particularmaleimide is resistant to ring opening. However, in unbuffered water,again at 25° C. and at a higher pH, the hydrolysis of mPEG2-MAL-40K wasdetermined to have a half-life of about 2.1 days, when measured in thesame way.

Example 3 Hydrolysis Rate Study of Polymer Succinimide Conjugates

The hydrolysis rates of representative protein and small molecule modelconjugates were investigated to examine the correlation between the ringopening tendencies of the polymer-terminated maleimides themselvesversus their conjugates.

Since large biomolecular components such as proteins have a dramaticeffect on the retention of conjugated molecules on common liquidchromatography columns, it is generally more difficult to measurekinetics of maleimide conjugates than it is for the polymers themselves.In this analysis, the open acid form of the maleamic acid was notdistinctly separable from the unopened or closed ring form. However, acombination analysis based upon size exclusion chromatography (HPLC-SE)and analytical protein electrophoresis (SDS-PAGE) was successfullyemployed to estimate the ring opening characteristics of polymericmaleimide protein conjugates, as well as conjugates prepared using modelnon-protein compounds.

In this study, two PEG-globular protein conjugates represented generallybelow were studied to examine their ring opening characteristics.

The top structure is a PEG-maleimide conjugate of Glob Protein 2, whereGlob Protein 2 is a protein having a molecular weight of approximately48 kDa. Glob Protein 2 was conjugated to a PEG maleimide derived from aPEG propionic acid, MW 30 kDa, which further included a medium-lengthlinker interposed between the propionic acid derived portion of thepolymer and the maleimide terminus. The linker in the top structure is—C(O)—NH(CH₂)₂—NH—C(O)—CH₂CH₂—.

The bottom structure is a PEG-maleimide conjugate of Glob Protein 1,where the protein possesses a molecular weight of about 11 kDa. Theconjugate was prepared using a linkerless maleimide (mPEG-MAL) having amolecular weight of about 20 kDa. The corresponding PEG maleimidestructure is 3-ET.

The bottom structure (Glob Protein 2) is completely ring opened after 24hours at pH 8.5 at room temperature, thus indicating the instability ofthis type of maleimidyl terminated polymer. Thus, this polymer conjugateis a good candidate for promoting the ring-opening reaction to provide achemically stable composition, that is to say, one at equilibrium, thatcomprises the polymer succinamic acid conjugate. Relative to thelinkerless form, however, the linker in the top structure (GlobProtein 1) appears to retard the ring opening, since the ring structurein the top conjugate is not completely ring-opened until 17 hours, at pH9, upon heating to 50° C. for 17 hours.

Example 4 Ring Opening Characteristics of Model PEG-SuccinimideConjugates

The hydrolysis rates of certain illustrative polymer maleimidesconjugated to a model compound, 2-mercaptoethanol, were determined toassess the tendency of the conjugates towards ring-opening, and thustheir suitability for the ring-opening approach provided herein.

Hydrolysis rate studies of conjugates having the structures shown below,where the linkers include portions designated as TRI, PEN, and MCH, wereconducted as described above for the unconjugated maleimides. Thehalf-lives shown were calculated from data taken at two different pHvalues. Similar to the unconjugated maleimides, the data indicate aslowing in reaction rate as the pHs drifted lower with increased ringopening. The linkage with the shortest hydrocarbon chain adjacent to thesuccinimide ring (i.e., TRI) was the fastest to open in comparison tothe other conjugates studied.

TABLE 3 Hydrolysis Half-lives of mPEG (5 k-Da) Maleimide Conjugates

Experimentally Determined Half-lives Linker, D pH 9.06 pH 8.11 TRI;trimethylene 31.4 hours 17.6 days PEN; pentamethylene — 28.5 days MCH;

43.3 hours —

Example 5 Hydrolysis at Various pH Values for a LinkerlessmPEG-Maleimide

Hydrolysis studies of conjugates formed by reaction of the modelcompound, 2-mercaptoethanol, with mPEG-5K-Maleimide were carried out asdescribed previously. A summary of the kinetics of the hydrolysisreaction of the conjugates at various pHs is provided in Table 4 below.

TABLE 4 HYDROLYSIS STUDY OF AN ADDUCT OF M-PEG(5K)-MAL WITH2-MERCAPTOETHANOL pH Half-life, min 12 <5 11 <15 10 30 9 600

Synthesis of an mPEG-5K-Maleimide Adduct with 2-Mercaptoethanol(mPEG-MAL-ME).

To a solution of mPEG(5000 Da)-maleimide (3.0 g, 0.0006 moles, Nektar,Huntsville, Ala.) in acetonitrile (60 ml), 2-mercaptoethanol (0.15 g,0.0190 moles) was added and the mixture was stirred overnight at roomtemperature under an argon atmosphere. The solvents were then distilledoff under reduced pressure. The residue was dissolved in dichloromethane(7.5 ml) and isopropyl alcohol was then added. The precipitated productwas filtered off and dried under reduced pressure. Yield: 2.80 g.

NMR (d6-DMSO): 2.78 ppm (bm, —S—CH ₂CH₂OH, 2H), 3.24 ppm (s, —OCH₃, 3H),3.51 ppm (s, PEG segment), 4.03 ppm (m, —CH—S—, 1H), 4.85 ppm (7, —OH,1H).

Hydrolysis at pH 9

mPEG-MAL-ME (0.2 g) was dissolved in 4 ml of distilled water and theresulting solution was added to 4 ml of 0.1 M phosphate buffer (pH=9.3).The pH was adjusted immediately to 9.0 by addition of 0.01M NaOH. 0.25ml samples of the solution were withdrawn at 1 h intervals and analyzedby HPLC. During measurement the pH of the solution was maintained withina range of 8.95-9.05 by periodic addition of 0.01M NaOH.

Isolation of Succinamic Acid Conjugates

Carrying out the hydrolysis reaction as described above, the productswere isolated from reactions conducted at pH 9 and pH 12. In each casethe products were the same. Two products of hydrolysis were formed, thecorresponding 2-position adduct and the 3-position adduct. Productassignments were made on the basis of spectral simulations. NMR analysisrevealed that the molar ratio of the 2-position adduct to the 3-positionadduct was 71 to 29.

Many modifications and other embodiments of the invention will come tomind to one skilled in the art to which this invention pertains havingthe benefit of teachings presented in the foregoing descriptions and theassociated drawings. Therefore, it is to be understood that theinvention is not to be limited to the specific embodiments disclosed andthat modifications and other embodiments are intended to be includedwithin the scope of the appended claims. Although specific terms areemployed herein, they are used in a generic and descriptive sense onlyand not for purposes of limitation.

1. A composition comprising a plurality of conjugates, each conjugate inthe plurality a protein derivatized with a water-soluble polymer,wherein the polymer is coupled to the protein via succinimide groupscovalently attached to either cysteine sulfhydryl groups or lysine aminogroups, and substantially all of the succinimide groups present in thecomposition are present in a ring-opened form.
 2. The composition ofclaim 1, wherein 95% or greater of all the succinimide groups present inthe composition are present in a ring-opened form.
 3. The composition ofclaim 1, wherein, the polymer is coupled to the protein via succinimidegroups covalently attached to cysteine sulfhydryl groups.
 4. Thecomposition of claim 3, wherein 95% or greater of all the succinimidegroups present in the composition are present in a ring-opened form. 5.The composition of claim 1, wherein, the polymer is coupled to theprotein via succinimide groups covalently attached to lysine aminogroups.
 6. The composition of claim 5, wherein 95% or greater of all thesuccinimide groups present in the composition are present in aring-opened form.
 7. The composition of claim 1, wherein thewater-soluble polymer is selected from the group consisting of apoly(alkylene oxide), poly(vinyl pyrrolidone), poly(vinyl alcohol),polyoxazoline, poly(acryloylmorpholine), and poly(oxyethylated polyol).8. The composition of claim 7, wherein the water-soluble polymer is apoly(alkylene oxide).
 9. The composition of claim 8, wherein thewater-soluble polymer is a poly(ethylene glycol).
 10. The composition ofclaim 9, wherein said poly(ethylene glycol) comprises an end-cappingmoiety.
 11. The composition of claim 10, wherein said end-capping moietyis selected from the group consisting alkoxy, substituted alkoxy,alkenyloxy, substituted alkenyloxy, alkynyloxy, substituted alkynyloxy,aryloxy, and substituted aryloxy.
 12. The composition of claim 11,wherein said end-capping moiety is selected from the group consisting ofmethoxy, ethoxy, and benzyloxy.
 13. The composition of claim 9, whereinsaid poly(ethylene glycol) has a nominal average molecular mass of fromabout 100 daltons to about 100,000 daltons.
 14. The composition of claim13, wherein said poly(ethylene glycol) has a nominal average molecularmass of from about 1,000 daltons to about 80,000 daltons.
 15. Thecomposition of claim 14, wherein said poly(ethylene glycol) has anominal average molecular mass of from about 2,000 daltons to about50,000 daltons.
 16. The composition of claim 9, wherein saidpoly(ethylene glycol) has a structure selected from the group consistingof linear, branched, forked, and multi-armed.
 17. The composition ofclaim 1, wherein said water-soluble polymer comprises a linker, L,interposed between said water-soluble polymer and said maleimide group.18. The composition of claim 9, wherein said poly(ethylene glycol) isdirectly attached to the nitrogen atom of said maleimide group.
 19. Thecomposition of claim 1, in the form of a precipitate.